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Oxygenation inhibits ovarian tumor growth by down-regulating STAT3 and cyclin-D1 expressions

Oxygenation inhibits ovarian tumor growth by down-regulating STAT3 and cyclin-D1 expressions Rese ARCh pApeR Cancer Biology & Therapy 10:4, 386-390; August 15, 2010; © 2010 Landes Bioscience Oxygenation inhibits ovarian tumor growth by downregulating STAT3 and cyclin-D1 expressions Karuppaiyah s elvendiran, M. Lakshmi Kuppusamy, s habnam Ahmed, Anna Bratasz, Guruguhan Meenakshisundaram, Brian K. Rivera, Mahmood Khan and periannan Kuppusamy Department of Internal Medicine and Comprehensive Cancer Center; Ohio s tate University; Columbus, Oh Us A Key words: oxygenation, ovarian cancer, hypoxia, STAT3, cyclin-D1 h ypoxia, which is commonly observed in many solid tumors, is a major impediment to chemo- or radiation therapy. h ypoxia is also known to overexpress/activate signal transducer and activator of transcription 3 (s TAT3) leading to tumor progression as well as drug resistance. We hypothesized that increased oxygenation of the hypoxic tumor may have an inhibitory effect on s TAT3 activation and hence tumor-growth inhibition. Mice containing human ovarian cancer xenograft tumor were exposed to hyperbaric oxygen (h BO; 100% oxygen; 2 atm; 90-min duration) daily, for up to 21 days. Mice exposed to h BO showed a significant reduction in tumor volume, with no effect on body weight. s TAT3 (Tyr 705) activation and cyclin-D1 protein/mRNA levels were significantly decreased up on h BO exposure. Interestingly, h BO exposure, in combination with weekly administration of cisplatin, also significantly reduced the tumor volume; however, this group of mice had drastically reduced body weight when compared to other groups. While conventional wisdom might suggest that increased oxygenation of tumors would promote tumor growth, the results of the present study indicated otherwise. h yperoxia appears to inhibit s TAT3 activation, which is a key step in the ovarian tumor progression. The study may have important implications for the treatment of ovarian cancer in the clinic. previously contraindicated in the treatment of malignancy due Introduction to the concern that hyperoxia may stimulate tumor growth via re-oxygenation of hypoxic tumor cells and increased neovas- Hypoxia plays a significant role in cancer therapy. It reduces the 1-3 15,16 cularization. sensitivity of tumors to radio- or chemotherapeutic agents. Repeated HBO exposures have been shown to Hence, there have been several approaches to oxygenate the induce angiogenesis leading to an increase in vascular density in hypoxic tumor before radiation or chemotherapy. One approach ischemic, necrotic and gangrenous tissues. Some recent stud- is to improve systemic oxygen delivery by increasing the dis- ies have reported an increase or early onset of metastases with solved oxygen concentration in plasma using special hemoglo- HBO therapy. Johnson et al. reported a tumor stimulatory effect bin preparations, peru fl orochemicals or hyperbaric oxygenation with HBO, with increased metastases in patients with cervical 4,5 18 (HBO). HBO treatment involves the administration of 100% cancer . This has been supported by other experimental studies oxygen under higher-than-atmospheric pressure. The additional and clinical trials. There are also other studies that showed a 19,20 pressure, coupled with inspiration of pure oxygen, substantially tumor inhibitory or negligible effect with HBO treatment. increases the amount of oxygen dissolved in the plasma. The On the other hand, a few studies have shown a suppressive effect 13,21,22 relatively high levels of oxygen deliverable under HBO exposure of HBO on tumor growth. make this approach attractive. Several studies in experimental Recently, we have reported that solid tumor xenografts of animal tumors have shown that HBO reduces the radioresistance ovarian cancer were severely hypoxic, with the partial pressure 6-9 23 of certain types of tumors and enhances the anticancer activ- of oxygen (pO ) about 2 mmHg. We observed that hypoxic 10,11 ity of doxorubicin and other alkylating agents. Clinical trials exposure of a human ovarian cancer cell line (A2780) in vitro have been initiated to study the effect of HBO inhalation on the resulted in a substantial increase in the STAT3 phosphoryla- radiotherapy for many types of malignant tumors. HBO expo- tion (Tyr705) which could be reversed within 12 hours after re- sure has been shown to increase tumor radiosensitivity in both exposure of the cells to normoxia. Because activation of STAT3 13,14 pre-clinical and clinical studies. directly regulates both cell-proliferation and survival genes that Although, HBO exposure has been shown to enhance the provide growth advantages to tumor cells, we hypothesized that radio- or chemosensitivity of tumor, the effect of HBO alone oxygenation of the ovarian tumor in vivo may have an attenuat- on the progression of malignancy is uncertain. HBO has been ing effect on STAT3 activation and hence inhibit tumor growth. *Correspondence to: Periannan Kuppusamy; Email: kuppusamy.1@osu.edu Submitted: 02/24/10; Revised: 05/20/10; Accepted: 05/21/10 Previously published online: www.landesbioscience.com/journals/cbt/article/12448 DOI: 10.4161/cbt.10.4.12448 386 Cancer Biology & Therapy Volume 10 Issue 4 Rese ARCh pApeR Rese ARCh pApeR volumes between the HBO- and cisplatin-treated groups. Nonparametric statistical analysis showed that there was a significant loss of body weight in the cisplatin treatment group when compared to the untreated group at days 12, 15 and 18 (p = 0.0079 for each of these time points). Significant differences in body weight were also observed between the cisplatin + HBO treatment group and the control group on days 5 and 10 (p = 0.0079 for these time points). No signifi - cant difference in body weight was found between the HBO-treated group and the control group (p > 0.3070 for all time points). The cisplatin + HBO group of mice were sacrificed prior to the planned conclusion of the study due to an extreme loss of body weight (Fig. 1C). These results suggested that HBO treatment alone was capable of inhibiting tumor growth, without any adverse effect on body weight. Effect of oxygenation on STAT3 and cyclin D1 expression. Tumor tissues excised from HBO-treated animals showed significantly reduced pSTAT3 (Tyr705) expression when compared to untreated controls (Fig. 2A). The treatments had no significant effect on the pSTAT3 (Ser727) or total STAT3 levels. We fur- ther observed that the expression of the cell-proliferation gene cyclin D1 was inhibited in HBO-treated animals (Fig. 2B). In addition, we confirmed through immuno - histochemical analysis that pSTAT3 levels were reduced in the HBO group (Fig. 2C). These results suggested that inhibition of ovarian tumor growth by HBO may be due to inhibition of pSTAT3 and cyclin D1 expressions. Discussion Figure 1. eff ect of h BO on the growth of A2780 ovarian xenograft tumor in mice. (A) picture of tumor-bearing mice shown at the end of the observation period. The present study has demonstrated the effect of hyper- Arrows indicate tumors. (B) Tumor volume. Both h BO and cisplatin-treated (cDDp) animals had significantly (*p < 0.05; N = 6 mice/group) reduced tumor baric oxygenation on the growth of a human ovar- volume when compared to untreated controls. (C) Body weight of the animals ian cancer xenograft tumor in mice. HBO exposure, during the treatment period. Cisplatin-treated animals had significantly (*p < administered daily for 21 days, significantly attenuated 0.05; N = 6 mice/group) reduced body weight when compared to untreated tumor growth, which correlated with the inhibition of controls and h BO-treated animals. # indicates a fourth group of animals, treated STAT3 activation and cyclin D1 expression. Recently, with both cisplatin and h BO that were euthanized on day 12 of the experimental period due to extreme loss of body weight. we reported that hypoxia (1% O ) stimulated STAT3 activation in human ovarian cancer cells (A2780) grown in vitro. We observed that activated STAT3 was more In the present study we evaluated the effect of HBO treatment on highly expressed than the traditional markers of hypoxia, HIF-1α the tumor growth and STAT3 activation in a murine xenograft or VEGF, in hypoxia-cultured ovarian cancer cells. This is note- model of human ovarian cancer. The results demonstrated that worthy, as the activation of STAT3 is a key factor in the develop- 24,25 HBO treatment resulted in the attenuation of STAT3 activation ment of cellular resistance to chemotherapy. The results of the leading to inhibition of tumor growth. present study show that STAT3 activation is modulated by tumor tissue oxygenation. Results In addition to STAT3 activation, it is likely that other signal- ing mechanisms could likewise be affected by a systemic increase Effect of oxygenation on tumor growth and body weight. in oxygen levels. The cell-proliferation gene cyclin D1 was down- Representative animals from each of the experimental groups regulated in HBO-treated animals. We did not observe any that survived to completion of the study are shown in Figure changes in HIF-1α or VEGF expression levels (data not shown). 1A. The HBO or cisplatin group of animals had significantly These results are consistent with previous reports from similar reduced tumor volume when compared to untreated group studies conducted on lung metastases, in which HBO had an (Fig. 1B). There was no significant difference in the tumor inhibitory effect on tumor growth. www.landesbioscience.com Cancer Biology & Therapy 387 We also used HBO as an adjuvant treatment in combination with the well-known ovarian cancer chemother- apy agent, cisplatin. This combination therapy significantly reduced the tumor volume within 2 weeks when compared with untreated animals and animals treated with HBO alone. However, this group of mice had significantly reduced body weight when compared to all other groups. At present, we do not know the precise etiology for this drastic weight loss observed in the combination treat- ment animals, and whether or not HBO treatment would enhance the systemic toxicity of cisplatin and/or cause addi- tional organ damage and failure. It has been postulated that HBO administra- tion promotes the formation of reactive oxygen species, thereby inducing oxida- tive stress within the tissues. In some studies, HBO has been shown to induce 28,29 oxidative injury, while others claim a 30-32 preventive effect. However, whether Figure 2. eff ect of oxygenation on ps TAT3 and Cyclin D1 expression. Western blot analyses were HBO acts as an oxidant promoter or as performed on tissues from excised A2780 tumor xenografts in untreated mice, and mice treated an antioxidant agent remains a matter with cisplatin (cDDp) or h BO. (A) Both treatments reduced the ps TAT3 (Tyr705; Y) and cyclin D1 levels, of debate. but not ps TAT3 (s er727, s ) or total s TAT3 levels. (B) Cyclin D1 and VeGF mRNA levels were analyzed HBO therapy has been investigated by RT-pCR. *p < 0.05 (N = 3). (C) Immunohistochemical staining showing ps TAT3. Arrows represent positively-stained areas. as a means by which to prevent cispl- 33,34 atin-mediated nephrotoxicity, and 35 34 otoxicity. Aydinoz et al. reported significant body-weight loss in cisplatin as well as cisplatin + ovarian cancer patients. Future studies may also include addi- HBO-treated rats. However, the most dramatic weight loss was tional investigation into other signaling mechanisms that may be observed in the cisplatin only-treated group. Atasoyu et al. responsible for HBO attenuation of ovarian tumor growth. have observed a high mortality rate in rats receiving cisplatin and cisplatin + HBO. Further, Yassuda et al. noted that 2 of 5 Materials and Methods guinea pigs treated with combination therapy died prior to the completion of the study. Thus far, the evidence suggests that Materials. Antibodies directed against pSTAT3 (Tyr705 and even under differing treatment conditions, combination therapy Ser727) were purchased from Cell Signaling Technolog y (Beverly, utilizing cisplatin and HBO should be avoided. The effect of MA). VEGF, cyclin D1, HIF-1α and STAT3 antibodies were HBO administration on tumor growth has also been contradic- purchased from Santa Cruz Biotechnology (Santa Cruz, CA). tory, with some studies reporting that HBO produces a growth- Cisplatin (cis-dichlorodiamine platinum, cDDP) was obtained stimulatory effect, while others report a growth-suppressive from Sigma. effect. Feldmeier et al. extensively reviewed experimental and Ovarian cancer xenograft tumors in mice and experimental clinical data and concluded that HBO exposure had no primary groups. A2780 human ovarian cancer cells (5 x 10 cells in 60 µ l or metastatic tumor stimulatory effect. of PBS) were subcutaneously injected in the back of 6-week-old Our investigation was intended as a simple pilot study on the BALB/c nude mice (National Cancer Institute). All treatments effect of HBO on ovarian tumor growth, primarily from a molec- were initiated on day 5, when the tumor size reached 2–4 mm. ular signaling standpoint. By re-oxygenating ovarian tumor The mice were divided into four groups, containing 8 mice per tissue, it is possible to decrease the levels of activated STAT3, group: (i) Control (untreated); (ii) HBO (only); (iii) Cisplatin thereby reducing the oncogenic effects affiliated with constitu- (only); (iv) HBO + Cisplatin (combination). HBO was adminis- tive activation of this oncogene. While combination therapy tered daily for 3 weeks. Cisplatin (100 mg/kg; i.p.) was adminis- using cisplatin may not be appealing, in light of our experimen- tered once per week for three weeks. tal results and those reported by others, we see an opportunity HBO protocol. Animals were placed in cages in a custom- to develop new chemotherapeutic agents that could be used in built small-animal hyperbaric chamber (PolyFab, Boston Plastics combination with HBO as an additional means of treatment for Manufacturing; Wilmington, MA). HBO (100% O ) was 388 Cancer Biology & Therapy Volume 10 Issue 4 administered at a pressure of 2 atm for 90 min. A minimum of immunou fl orescence staining, 8- µ m-thick tissue sections were 15-min pressurization and depressurization was allowed for the serially rehydrated in 100%, 95% and 80% ethanol after depa- animals to adjust to the changes in pressure. HBO was adminis- raffinization with xylene. Slides were kept in steam for 30 min tered daily, for up to 21 days. The tumor size was measured twice and then washed in PBS (pH 7.4) three times for 5 min each. The per week using a digital vernier caliper and the tumor volume tissue sections were first incubated with 2% goat serum and 5% was calculated from the orthogonal dimensions (d , d , d ) using bovine serum albumin in PBS (to reduce nonspecific binding), 1 2 3 the formula (d x d x d ) x π/6. The animals were euthanized at and then by incubation for 4 h with an anti-mouse anti-pSTAT3, 1 2 3 the end of the experimental period. Tumor tissues were collected followed by incubation with secondary antibodies (1:1,000 and subjected to western blotting and immunohistochemical dilutions) conjugated to horseradish peroxidase (HRP)-labeled analyses. sheep anti-mouse IgG or HRP-labeled donkey anti-rabbit IgG Immunoblot analysis. The xenografted tumor tissue samples (Amersham Pharmacia Biotech). The tissue slides were visualized were homogenized on ice and the tissue lysates were prepared using a Nikon fluorescence microscope. in nondenaturing lysis buffer containing 10-mM Tris-HCl (pH Reverse-transcription PCR (RT-PCR). Total RNA isolated 7.4), 150-mM NaCl, 1% Triton X-100, 1-mM EDTA, 1-mM from ovarian tumor tissue was prepared with TRIzol (Life EGTA, 0.3-mM phenylmethylsulfonyl fluoride, 0.2-mM sodium Technologies, Grand Island, New York) according to the manu- orthovanadate, 0.5% NP40, 1-µ g/ml aprotonin and 1-µ g/ml facturer’s instructions. RNA quantification was done using spec - leupetin. The tissues lysates were centrifuged at 10,000 xg for trophotometry. Reverse transcription RT-PCR analysis for the 20 min at 4°C, and then supernatant was separated. The pro- mRNA expressions in cyclin D1, VEGF and the internal control tein concentration in the lysates was determined using a Pierce GAPDH was carried out using a GeneAmp PCR System Veriti detergent-compatible protein assay kit. For western blotting, 25 thermocycler (Applied Biosystems, Foster City, CA) under the to 50 µ g of protein lysate per sample was denatured in 2x SDS- following conditions: initial denaturation at 94°C for 2 min, 35 PAGE sample buffer and subjected to SDS-PAGE on a 10% or cycles of amplification (denaturation at 94°C for 30 s, annealing 12.5% tris-glycine gel. The separated proteins were transferred at 50°C for 30 s and extension at 72°C for 30 s), and extension to a PVDF membrane and the membrane was blocked with 5% at 72°C for 5 min. The PCR products were electrophoresed on nonfat milk powder (w/v) in TBST containing 10-mM Tris, 1.5% agarose gel and stained with ethidium bromide. 100-mM NaCl and 0.1% Tween 20 for 1 h at room temperature, Statistical analysis. Due to the small sample size, non- or overnight at 4°C. The membranes were then incubated with parametric Wilcoxon rank sum tests were performed for paired the primary antibodies mentioned previously. The bound anti- comparisons. Bonferroni corrections were used to adjust the sig- bodies were detected with horseradish peroxidase (HRP)-labeled nificance level for multiple comparisons. A p-value of less than sheep anti-mouse IgG or HRP-labeled donkey anti-rabbit IgG 0.0083 (=0.05/6) was considered significant with Bonferroni (Amersham Pharmacia Biotech) using an enhanced chemilumi- corrections for the non-parametric tests. Otherwise, a p value of nescence detection system (ECL Advanced Kit). Protein expres- less than 0.05 was considered significant. sions were quantified using Image Gauge (version 3.45) software. Acknowledgements Immunohistochemistry. Tumor tissues were fixed in forma - lin and embedded in paraffin. Six-micron-thick tissue sections We acknowledge the financial support from the Kaleidoscope of were obtained and used for hematoxylin and eosin staining. For Hope Foundation for Ovarian Cancer Research. 7. Tang H, Sun Y, Xu C, Zhou T, Gao X, Wang L. 13. Bennett M, Feldmeier J, Smee R, Milross C. Hyperbaric References Effects of hyperbaric oxygen therapy on tumor growth oxygenation for tumour sensitisation to radiotherapy. 1. Rockwell S, Dobrucki IT, Kim EY, Marrison ST, Vu in murine model of PC-3 prostate cancer cell line. Cochrane Database Syst Rev 2005; 005007. VT. Hypoxia and radiation therapy: past history, ongo- Urology 2009; 73:205-8. 14. Teguh DN, Levendag PC, Noever I, Voet P, van der ing research and future promise. Curr Mol Med 2009; 8. Bennett M, Feldmeier J, Smee R, Milross C. Hyperbaric Est H, van Rooij P, et al. Early Hyperbaric Oxygen 9:442-58. oxygenation for tumour sensitisation to radiotherapy: a Therapy for Reducing Radiotherapy Side Effects: Early 2. Huerta S, Gao X, Saha D. Mechanisms of resistance systematic review of randomised controlled trials. Results of a Randomized Trial in Oropharyngeal and to ionizing radiation in rectal cancer. Expert Rev Mol Cancer Treat Rev 2008; 34:577-91. Nasopharyngeal Cancer. Int J Radiat Oncol Biol Phys Diagn 2009; 9:469-80. 2009; 75:711-6. 9. Yoshida T, Kawashima A, Ujike T, Uemura M, 3. Ruan K, Song G, Ouyang G. Role of hypoxia in the Nishimura K, Miyoshi S. Hyperbaric oxygen therapy 15. Feldmeier J, Carl U, Hartmann K, Sminia P. Hyperbaric hallmarks of human cancer. J Cell Biochem 2009; for radiation-induced hemorrhagic cystitis. Int J Urol oxygen: does it promote growth or recurrence of malig- 107:1053-62. 2008; 15:639-41. nancy? Undersea Hyperb Med 2003; 30:1-18. 4. Daruwalla J, Christophi C. The effect of hyperbaric 10. Kalns J, Krock L, Piepmeier E Jr. The effect of hyper- 16. Moon RE, Feldmeier JJ. Hyperbaric oxygen: an evi- oxygen therapy on tumour growth in a mouse model baric oxygen on growth and chemosensitivity of meta- dence based approach to its application. Undersea of colorectal cancer liver metastases. Eur J Cancer 2006; static prostate cancer. Anticancer Res 1998; 18:363-7. Hyperb Med 2002; 29:1-3. 42:3304-11. 11. Daruwalla J, Greish K, Nikfarjam M, Millar 17. Marx RE, Ehler WJ, Tayapongsak P, Pierce LW. 5. Daruwalla J, Christophi C. Hyperbaric oxygen therapy for I, Malcontenti-Wilson C, Iyer AK, Christophi C. Relationship of oxygen dose to angiogenesis induction malignancy: a review. World J Surg 2006; 30:2112-31. Evaluation of the effect of SMA-pirarubicin micelles in irradiated tissue. Am J Surg 1990; 160:519-24. 6. Alagoz T, Buller RE, Anderson B, Terrell KL, Squatrito on colorectal cancer liver metastases and of hyperbaric 18. Johnson RJ, Wiseman N, Lauchlan SC. The effect of RC, Niemann TH, et al. Evaluation of hyperbaric oxy- oxygen in CBA mice. J Drug Target 2007; 15:487-95. hyperbaric oxygen on tumour metastases in mice. Clin gen as a chemosensitizer in the treatment of epithelial 12. Mayer R, Hamilton-Farrell MR, van der Kleij AJ, Radiol 1971; 22:538-40. ovarian cancer in xenografts in mice. Cancer 1995; Schmutz J, Granstrom G, Sicko Z, et al. Hyperbaric 75:2313-22. oxygen and radiotherapy. Strahlenther Onkol 2005; 181:113-23. www.landesbioscience.com Cancer Biology & Therapy 389 19. Dische S, Saunders MI, Sealy R, Werner ID, Verma N, 24. Gritsko T, Williams A, Turkson J, Kaneko S, Bowman 30. Yasar M, Yildiz S, Mas R, Dundar K, Yildirim A, Foy C, Bentzen SM. Carcinoma of the cervix and the T, Huang M, et al. Persistent activation of stat3 sig- Korkmaz A, et al. The effect of hyperbaric oxygen use of hyperbaric oxygen with radiotherapy: a report of naling induces survivin gene expression and confers treatment on oxidative stress in experimental acute a randomised controlled trial. Radiother Oncol 1999; resistance to apoptosis in human breast cancer cells. necrotizing pancreatitis. Physiol Res 2003; 52:111-6. 53:93-8. Clin Cancer Res 2006; 12:11-9. 31. Gulec B, Yasar M, Yildiz S, Oter S, Akay C, Deveci S, 20. Granstrom G, Tjellstrom A, Branemark PI. 25. Lo HW, Cao X, Zhu H, Ali-Osman F. Constitutively Sen D. Effect of hyperbaric oxygen on experimental Osseointegrated implants in irradiated bone: a case- activated STAT3 frequently coexpresses with epidermal acute distal colitis. Physiol Res 2004; 53:493-9. controlled study using adjunctive hyperbaric oxygen growth factor receptor in high-grade gliomas and tar- 32. Ozden TA, Uzun H, Bohloli M, Toklu AS, Paksoy therapy. J Oral Maxillofac Surg 1999; 57:493-9. geting STAT3 sensitizes them to Iressa and alkylators. M, Simsek G, et al. The effects of hyperbaric oxygen Clin Cancer Res 2008; 14:6042-54. 21. Akiya T, Nakada T, Katayama T, Ota K, Chikenji M, treatment on oxidant and antioxidants levels during Matsushita T, Saito H. Hyperbaric oxygenation for 26. Sklizovic D, Sanger JR, Kindwall EP, Fink JG, Grunert liver regeneration in rats. Tohoku J Exp Med 2004; experimental bladder tumor II. Hyperbaric oxygen- BK, Campbell BH. Hyperbaric oxygen therapy and 203:253-65. ation in combination with chemotherapy in N-butyl- squamous cell carcinoma cell line growth. Head Neck 33. Atasoyu EM, Yildiz S, Bilgi O, Cermik H, Evrenkaya N-(4-hydroxybutyl)nitrosamine-induced bladder 1993; 15:236-40. R, Aktas S, et al. Investigation of the role of hyperbaric tumors. Eur Urol 1988; 14:150-5. 27. Jamieson D. Oxygen toxicity and reactive oxygen oxygen therapy in cisplatin-induced nephrotoxicity in 22. Gerlach NL, Barkhuysen R, Kaanders JH, Janssens metabolites in mammals. Free Radic Biol Med 1989; rats. Arch Toxicol 2005; 79:289-93. GO, Sterk W, Merkx MA. The effect of hyperbaric oxy- 7:87-108. 34. Aydinoz S, Uzun G, Cermik H, Atasoyu EM, Yildiz S, gen therapy on quality of life in oral and oropharyngeal 28. Benedetti S, Lamorgese A, Piersantelli M, Pagliarani S, Karagoz B, Evrenkaya R. Effects of different doses of cancer patients treated with radiotherapy. Int J Oral Benvenuti F, Canestrari F. Oxidative stress and antioxi- hyperbaric oxygen on cisplatin-induced nephrotoxicity. Maxillofac Surg 2008; 37:255-9. dant status in patients undergoing prolonged exposure Ren Fail 2007; 29:257-63. 23. Selvendiran K, Bratasz A, Kuppusamy ML, Tazi MF, to hyperbaric oxygen. Clin Biochem 2004; 37:312-7. 35. Yassuda CC, Righetti AE, Cury MC, Hyppolito MA, Rivera BK, Kuppusamy P. Hypoxia induces chemore- 29. Oter S, Korkmaz A, Topal T, Ozcan O, Sadir S, Ozler M, Oliveira JA, Feres O. The role of hyperbaric oxygen sistance in ovarian cancer cells by activation of signal et al. Correlation between hyperbaric oxygen exposure therapy (hot) as an otoprotection agent against cisplatin transducer and activator of transcription 3. Int J Cancer pressures and oxidative parameters in rat lung, brain and ototoxicity. Acta Cir Bras 2008; 23:72-6. 2009; 125:2198-204. erythrocytes. Clin Biochem 2005; 38:706-11. 390 Cancer Biology & Therapy Volume 10 Issue 4 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

Oxygenation inhibits ovarian tumor growth by down-regulating STAT3 and cyclin-D1 expressions

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Abstract

Rese ARCh pApeR Cancer Biology & Therapy 10:4, 386-390; August 15, 2010; © 2010 Landes Bioscience Oxygenation inhibits ovarian tumor growth by downregulating STAT3 and cyclin-D1 expressions Karuppaiyah s elvendiran, M. Lakshmi Kuppusamy, s habnam Ahmed, Anna Bratasz, Guruguhan Meenakshisundaram, Brian K. Rivera, Mahmood Khan and periannan Kuppusamy Department of Internal Medicine and Comprehensive Cancer Center; Ohio s tate University; Columbus, Oh Us A Key words: oxygenation, ovarian cancer, hypoxia, STAT3, cyclin-D1 h ypoxia, which is commonly observed in many solid tumors, is a major impediment to chemo- or radiation therapy. h ypoxia is also known to overexpress/activate signal transducer and activator of transcription 3 (s TAT3) leading to tumor progression as well as drug resistance. We hypothesized that increased oxygenation of the hypoxic tumor may have an inhibitory effect on s TAT3 activation and hence tumor-growth inhibition. Mice containing human ovarian cancer xenograft tumor were exposed to hyperbaric oxygen (h BO; 100% oxygen; 2 atm; 90-min duration) daily, for up to 21 days. Mice exposed to h BO showed a significant reduction in tumor volume, with no effect on body weight. s TAT3 (Tyr 705) activation and cyclin-D1 protein/mRNA levels were significantly decreased up on h BO exposure. Interestingly, h BO exposure, in combination with weekly administration of cisplatin, also significantly reduced the tumor volume; however, this group of mice had drastically reduced body weight when compared to other groups. While conventional wisdom might suggest that increased oxygenation of tumors would promote tumor growth, the results of the present study indicated otherwise. h yperoxia appears to inhibit s TAT3 activation, which is a key step in the ovarian tumor progression. The study may have important implications for the treatment of ovarian cancer in the clinic. previously contraindicated in the treatment of malignancy due Introduction to the concern that hyperoxia may stimulate tumor growth via re-oxygenation of hypoxic tumor cells and increased neovas- Hypoxia plays a significant role in cancer therapy. It reduces the 1-3 15,16 cularization. sensitivity of tumors to radio- or chemotherapeutic agents. Repeated HBO exposures have been shown to Hence, there have been several approaches to oxygenate the induce angiogenesis leading to an increase in vascular density in hypoxic tumor before radiation or chemotherapy. One approach ischemic, necrotic and gangrenous tissues. Some recent stud- is to improve systemic oxygen delivery by increasing the dis- ies have reported an increase or early onset of metastases with solved oxygen concentration in plasma using special hemoglo- HBO therapy. Johnson et al. reported a tumor stimulatory effect bin preparations, peru fl orochemicals or hyperbaric oxygenation with HBO, with increased metastases in patients with cervical 4,5 18 (HBO). HBO treatment involves the administration of 100% cancer . This has been supported by other experimental studies oxygen under higher-than-atmospheric pressure. The additional and clinical trials. There are also other studies that showed a 19,20 pressure, coupled with inspiration of pure oxygen, substantially tumor inhibitory or negligible effect with HBO treatment. increases the amount of oxygen dissolved in the plasma. The On the other hand, a few studies have shown a suppressive effect 13,21,22 relatively high levels of oxygen deliverable under HBO exposure of HBO on tumor growth. make this approach attractive. Several studies in experimental Recently, we have reported that solid tumor xenografts of animal tumors have shown that HBO reduces the radioresistance ovarian cancer were severely hypoxic, with the partial pressure 6-9 23 of certain types of tumors and enhances the anticancer activ- of oxygen (pO ) about 2 mmHg. We observed that hypoxic 10,11 ity of doxorubicin and other alkylating agents. Clinical trials exposure of a human ovarian cancer cell line (A2780) in vitro have been initiated to study the effect of HBO inhalation on the resulted in a substantial increase in the STAT3 phosphoryla- radiotherapy for many types of malignant tumors. HBO expo- tion (Tyr705) which could be reversed within 12 hours after re- sure has been shown to increase tumor radiosensitivity in both exposure of the cells to normoxia. Because activation of STAT3 13,14 pre-clinical and clinical studies. directly regulates both cell-proliferation and survival genes that Although, HBO exposure has been shown to enhance the provide growth advantages to tumor cells, we hypothesized that radio- or chemosensitivity of tumor, the effect of HBO alone oxygenation of the ovarian tumor in vivo may have an attenuat- on the progression of malignancy is uncertain. HBO has been ing effect on STAT3 activation and hence inhibit tumor growth. *Correspondence to: Periannan Kuppusamy; Email: kuppusamy.1@osu.edu Submitted: 02/24/10; Revised: 05/20/10; Accepted: 05/21/10 Previously published online: www.landesbioscience.com/journals/cbt/article/12448 DOI: 10.4161/cbt.10.4.12448 386 Cancer Biology & Therapy Volume 10 Issue 4 Rese ARCh pApeR Rese ARCh pApeR volumes between the HBO- and cisplatin-treated groups. Nonparametric statistical analysis showed that there was a significant loss of body weight in the cisplatin treatment group when compared to the untreated group at days 12, 15 and 18 (p = 0.0079 for each of these time points). Significant differences in body weight were also observed between the cisplatin + HBO treatment group and the control group on days 5 and 10 (p = 0.0079 for these time points). No signifi - cant difference in body weight was found between the HBO-treated group and the control group (p > 0.3070 for all time points). The cisplatin + HBO group of mice were sacrificed prior to the planned conclusion of the study due to an extreme loss of body weight (Fig. 1C). These results suggested that HBO treatment alone was capable of inhibiting tumor growth, without any adverse effect on body weight. Effect of oxygenation on STAT3 and cyclin D1 expression. Tumor tissues excised from HBO-treated animals showed significantly reduced pSTAT3 (Tyr705) expression when compared to untreated controls (Fig. 2A). The treatments had no significant effect on the pSTAT3 (Ser727) or total STAT3 levels. We fur- ther observed that the expression of the cell-proliferation gene cyclin D1 was inhibited in HBO-treated animals (Fig. 2B). In addition, we confirmed through immuno - histochemical analysis that pSTAT3 levels were reduced in the HBO group (Fig. 2C). These results suggested that inhibition of ovarian tumor growth by HBO may be due to inhibition of pSTAT3 and cyclin D1 expressions. Discussion Figure 1. eff ect of h BO on the growth of A2780 ovarian xenograft tumor in mice. (A) picture of tumor-bearing mice shown at the end of the observation period. The present study has demonstrated the effect of hyper- Arrows indicate tumors. (B) Tumor volume. Both h BO and cisplatin-treated (cDDp) animals had significantly (*p < 0.05; N = 6 mice/group) reduced tumor baric oxygenation on the growth of a human ovar- volume when compared to untreated controls. (C) Body weight of the animals ian cancer xenograft tumor in mice. HBO exposure, during the treatment period. Cisplatin-treated animals had significantly (*p < administered daily for 21 days, significantly attenuated 0.05; N = 6 mice/group) reduced body weight when compared to untreated tumor growth, which correlated with the inhibition of controls and h BO-treated animals. # indicates a fourth group of animals, treated STAT3 activation and cyclin D1 expression. Recently, with both cisplatin and h BO that were euthanized on day 12 of the experimental period due to extreme loss of body weight. we reported that hypoxia (1% O ) stimulated STAT3 activation in human ovarian cancer cells (A2780) grown in vitro. We observed that activated STAT3 was more In the present study we evaluated the effect of HBO treatment on highly expressed than the traditional markers of hypoxia, HIF-1α the tumor growth and STAT3 activation in a murine xenograft or VEGF, in hypoxia-cultured ovarian cancer cells. This is note- model of human ovarian cancer. The results demonstrated that worthy, as the activation of STAT3 is a key factor in the develop- 24,25 HBO treatment resulted in the attenuation of STAT3 activation ment of cellular resistance to chemotherapy. The results of the leading to inhibition of tumor growth. present study show that STAT3 activation is modulated by tumor tissue oxygenation. Results In addition to STAT3 activation, it is likely that other signal- ing mechanisms could likewise be affected by a systemic increase Effect of oxygenation on tumor growth and body weight. in oxygen levels. The cell-proliferation gene cyclin D1 was down- Representative animals from each of the experimental groups regulated in HBO-treated animals. We did not observe any that survived to completion of the study are shown in Figure changes in HIF-1α or VEGF expression levels (data not shown). 1A. The HBO or cisplatin group of animals had significantly These results are consistent with previous reports from similar reduced tumor volume when compared to untreated group studies conducted on lung metastases, in which HBO had an (Fig. 1B). There was no significant difference in the tumor inhibitory effect on tumor growth. www.landesbioscience.com Cancer Biology & Therapy 387 We also used HBO as an adjuvant treatment in combination with the well-known ovarian cancer chemother- apy agent, cisplatin. This combination therapy significantly reduced the tumor volume within 2 weeks when compared with untreated animals and animals treated with HBO alone. However, this group of mice had significantly reduced body weight when compared to all other groups. At present, we do not know the precise etiology for this drastic weight loss observed in the combination treat- ment animals, and whether or not HBO treatment would enhance the systemic toxicity of cisplatin and/or cause addi- tional organ damage and failure. It has been postulated that HBO administra- tion promotes the formation of reactive oxygen species, thereby inducing oxida- tive stress within the tissues. In some studies, HBO has been shown to induce 28,29 oxidative injury, while others claim a 30-32 preventive effect. However, whether Figure 2. eff ect of oxygenation on ps TAT3 and Cyclin D1 expression. Western blot analyses were HBO acts as an oxidant promoter or as performed on tissues from excised A2780 tumor xenografts in untreated mice, and mice treated an antioxidant agent remains a matter with cisplatin (cDDp) or h BO. (A) Both treatments reduced the ps TAT3 (Tyr705; Y) and cyclin D1 levels, of debate. but not ps TAT3 (s er727, s ) or total s TAT3 levels. (B) Cyclin D1 and VeGF mRNA levels were analyzed HBO therapy has been investigated by RT-pCR. *p < 0.05 (N = 3). (C) Immunohistochemical staining showing ps TAT3. Arrows represent positively-stained areas. as a means by which to prevent cispl- 33,34 atin-mediated nephrotoxicity, and 35 34 otoxicity. Aydinoz et al. reported significant body-weight loss in cisplatin as well as cisplatin + ovarian cancer patients. Future studies may also include addi- HBO-treated rats. However, the most dramatic weight loss was tional investigation into other signaling mechanisms that may be observed in the cisplatin only-treated group. Atasoyu et al. responsible for HBO attenuation of ovarian tumor growth. have observed a high mortality rate in rats receiving cisplatin and cisplatin + HBO. Further, Yassuda et al. noted that 2 of 5 Materials and Methods guinea pigs treated with combination therapy died prior to the completion of the study. Thus far, the evidence suggests that Materials. Antibodies directed against pSTAT3 (Tyr705 and even under differing treatment conditions, combination therapy Ser727) were purchased from Cell Signaling Technolog y (Beverly, utilizing cisplatin and HBO should be avoided. The effect of MA). VEGF, cyclin D1, HIF-1α and STAT3 antibodies were HBO administration on tumor growth has also been contradic- purchased from Santa Cruz Biotechnology (Santa Cruz, CA). tory, with some studies reporting that HBO produces a growth- Cisplatin (cis-dichlorodiamine platinum, cDDP) was obtained stimulatory effect, while others report a growth-suppressive from Sigma. effect. Feldmeier et al. extensively reviewed experimental and Ovarian cancer xenograft tumors in mice and experimental clinical data and concluded that HBO exposure had no primary groups. A2780 human ovarian cancer cells (5 x 10 cells in 60 µ l or metastatic tumor stimulatory effect. of PBS) were subcutaneously injected in the back of 6-week-old Our investigation was intended as a simple pilot study on the BALB/c nude mice (National Cancer Institute). All treatments effect of HBO on ovarian tumor growth, primarily from a molec- were initiated on day 5, when the tumor size reached 2–4 mm. ular signaling standpoint. By re-oxygenating ovarian tumor The mice were divided into four groups, containing 8 mice per tissue, it is possible to decrease the levels of activated STAT3, group: (i) Control (untreated); (ii) HBO (only); (iii) Cisplatin thereby reducing the oncogenic effects affiliated with constitu- (only); (iv) HBO + Cisplatin (combination). HBO was adminis- tive activation of this oncogene. While combination therapy tered daily for 3 weeks. Cisplatin (100 mg/kg; i.p.) was adminis- using cisplatin may not be appealing, in light of our experimen- tered once per week for three weeks. tal results and those reported by others, we see an opportunity HBO protocol. Animals were placed in cages in a custom- to develop new chemotherapeutic agents that could be used in built small-animal hyperbaric chamber (PolyFab, Boston Plastics combination with HBO as an additional means of treatment for Manufacturing; Wilmington, MA). HBO (100% O ) was 388 Cancer Biology & Therapy Volume 10 Issue 4 administered at a pressure of 2 atm for 90 min. A minimum of immunou fl orescence staining, 8- µ m-thick tissue sections were 15-min pressurization and depressurization was allowed for the serially rehydrated in 100%, 95% and 80% ethanol after depa- animals to adjust to the changes in pressure. HBO was adminis- raffinization with xylene. Slides were kept in steam for 30 min tered daily, for up to 21 days. The tumor size was measured twice and then washed in PBS (pH 7.4) three times for 5 min each. The per week using a digital vernier caliper and the tumor volume tissue sections were first incubated with 2% goat serum and 5% was calculated from the orthogonal dimensions (d , d , d ) using bovine serum albumin in PBS (to reduce nonspecific binding), 1 2 3 the formula (d x d x d ) x π/6. The animals were euthanized at and then by incubation for 4 h with an anti-mouse anti-pSTAT3, 1 2 3 the end of the experimental period. Tumor tissues were collected followed by incubation with secondary antibodies (1:1,000 and subjected to western blotting and immunohistochemical dilutions) conjugated to horseradish peroxidase (HRP)-labeled analyses. sheep anti-mouse IgG or HRP-labeled donkey anti-rabbit IgG Immunoblot analysis. The xenografted tumor tissue samples (Amersham Pharmacia Biotech). The tissue slides were visualized were homogenized on ice and the tissue lysates were prepared using a Nikon fluorescence microscope. in nondenaturing lysis buffer containing 10-mM Tris-HCl (pH Reverse-transcription PCR (RT-PCR). Total RNA isolated 7.4), 150-mM NaCl, 1% Triton X-100, 1-mM EDTA, 1-mM from ovarian tumor tissue was prepared with TRIzol (Life EGTA, 0.3-mM phenylmethylsulfonyl fluoride, 0.2-mM sodium Technologies, Grand Island, New York) according to the manu- orthovanadate, 0.5% NP40, 1-µ g/ml aprotonin and 1-µ g/ml facturer’s instructions. RNA quantification was done using spec - leupetin. The tissues lysates were centrifuged at 10,000 xg for trophotometry. Reverse transcription RT-PCR analysis for the 20 min at 4°C, and then supernatant was separated. The pro- mRNA expressions in cyclin D1, VEGF and the internal control tein concentration in the lysates was determined using a Pierce GAPDH was carried out using a GeneAmp PCR System Veriti detergent-compatible protein assay kit. For western blotting, 25 thermocycler (Applied Biosystems, Foster City, CA) under the to 50 µ g of protein lysate per sample was denatured in 2x SDS- following conditions: initial denaturation at 94°C for 2 min, 35 PAGE sample buffer and subjected to SDS-PAGE on a 10% or cycles of amplification (denaturation at 94°C for 30 s, annealing 12.5% tris-glycine gel. The separated proteins were transferred at 50°C for 30 s and extension at 72°C for 30 s), and extension to a PVDF membrane and the membrane was blocked with 5% at 72°C for 5 min. The PCR products were electrophoresed on nonfat milk powder (w/v) in TBST containing 10-mM Tris, 1.5% agarose gel and stained with ethidium bromide. 100-mM NaCl and 0.1% Tween 20 for 1 h at room temperature, Statistical analysis. Due to the small sample size, non- or overnight at 4°C. The membranes were then incubated with parametric Wilcoxon rank sum tests were performed for paired the primary antibodies mentioned previously. The bound anti- comparisons. Bonferroni corrections were used to adjust the sig- bodies were detected with horseradish peroxidase (HRP)-labeled nificance level for multiple comparisons. A p-value of less than sheep anti-mouse IgG or HRP-labeled donkey anti-rabbit IgG 0.0083 (=0.05/6) was considered significant with Bonferroni (Amersham Pharmacia Biotech) using an enhanced chemilumi- corrections for the non-parametric tests. Otherwise, a p value of nescence detection system (ECL Advanced Kit). Protein expres- less than 0.05 was considered significant. sions were quantified using Image Gauge (version 3.45) software. Acknowledgements Immunohistochemistry. Tumor tissues were fixed in forma - lin and embedded in paraffin. Six-micron-thick tissue sections We acknowledge the financial support from the Kaleidoscope of were obtained and used for hematoxylin and eosin staining. For Hope Foundation for Ovarian Cancer Research. 7. Tang H, Sun Y, Xu C, Zhou T, Gao X, Wang L. 13. Bennett M, Feldmeier J, Smee R, Milross C. Hyperbaric References Effects of hyperbaric oxygen therapy on tumor growth oxygenation for tumour sensitisation to radiotherapy. 1. Rockwell S, Dobrucki IT, Kim EY, Marrison ST, Vu in murine model of PC-3 prostate cancer cell line. Cochrane Database Syst Rev 2005; 005007. VT. Hypoxia and radiation therapy: past history, ongo- Urology 2009; 73:205-8. 14. Teguh DN, Levendag PC, Noever I, Voet P, van der ing research and future promise. Curr Mol Med 2009; 8. 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Cancer Biology & TherapyTaylor & Francis

Published: Aug 15, 2010

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