Cellular response to chemotherapy and radiation in cervical cancer

Transcription

1 American Journal of Obstetrics and Gynecology (2005) 192, Cellular response to chemotherapy and radiation in cervical cancer Angela Saxena, MD, Catheryn Yashar, MD, Douglas D. Taylor, PhD, Cicek Gercel-Taylor, PhD* Department of Obstetrics, Gynecology and Women s Health and Department of Radiation Oncology, University of Louisville School of Medicine, Louisville, Ky Received for publication September 1, 2004; accepted December 9, 2004 KEY WORDS Cervical cancer Radiotherapy Cisplatin Paclitaxel 5-fluorouracil Effect of irradiation alone and irradiation after 5-fluorouracil (5-FU), paclitaxel, or cisplatin (CDDP) was investigated in human cervical cell lines (CaSki, ME180, SiHa, and C33A). Highrisk human papillomavirus (HPV) (C) CaSki and SiHa cells were the most resistant to CDDP, 5-FU, and radiation treatments. Radiation and CDDP and 5-FU resulted in decreased survival of HPV 16 and 18 (C) cells, whereas addition of paclitaxel to radiation treatments decreased killing. Enhanced killing of ME180 cells containing HPV39 sequences was demonstrated with chemoradiotherapy with all agents. HPV( ) C33A was more sensitive to radiation than the other cell lines, and the addition of chemotherapeutic agents did not result in significant change in cytotoxicity. Expression of survivin was inversely proportional to cell sensitivity to CDDP, 5-FU, and radiation. Constitutive AKT levels are the lowest in cell lines that are the most resistant to CDDP, 5-FU, and radiation. These data provide correlation of response to combined therapeutic modalities with HPV status of cervical cancer and expression of survivin and AKT. Ó 2005 Elsevier Inc. All rights reserved. Cervical cancer is one of the major causes of cancer death worldwide. Particular human papillomavirus (HPV) types are carcinogenic in humans and more than 99% of cervical cancers are initiated by HPV infection. Genital HPV types are divided into high-, medium-, and low-risk groups based on the frequency of association with malignant tumors. 1 * Reprint requests: Cicek Gercel-Taylor, PhD, University of Louisville, School of Medicine, Department of Obstetrics, Gynecology and Women s Health, 511 S Floyd St, Room 416, Louisville, KY cgtay101@gwise.louisville.edu Treatment of invasive cancer involves management of the primary lesion and sites of metastatic disease. Surgery and radiation therapy may be used for primary treatment, although definitive surgery is usually limited to patients with stages I or early IIa disease. Currently chemoradiotherapy involving cisplatin (CDDP) and 5- fluorouracil (5-FU) is the standard of care. 2 However, improvement in the treatment of locally advanced disease is needed. Resistance of cancer to radiotherapy is a primary cause of treatment failure. Drug combinations eliminating radiation or CDDP-resistant cells may improve local control rates. 3 The radiosensitivity of tumor cells is measured in vitro by clonogenic survival. 4 Because this assay is time /$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: /j.ajog

2 1400 Saxena et al Figure 1 A-C, Chemosensitivity of cervical cancer cells (C33A, CaSki, ME180, SiHa) cells to CDDP, 5-FU, and Tax. SRB assay was used to generate the survival curves with assays run in quadruplicate twice. Results are shown as mean G SEM. and resources consuming, there is interest in assessing nonclonogenic endpoints for measuring tumor radiosensitivity and individualization of treatment. 5 This study investigates an association between HPV status of cervical cancer cells and their sensitivity to chemotherapeutic agents (5-FU, paclitaxel [Tax], CDDP), and radiation. Constitutive expression of p53, p73, survivin, mcl-1, and AKT was studied in these cell lines. Material and methods Cell lines CaSki (HPV-16, 18), C33A (HPV negative), SiHa (HPV-18), and ME180 (HPV-39) cells were used. All cell lines were cultured in RPMI medium supplemented with charcoal-treated 10% fetal bovine serum, at 37(C in a humidified CO 2 incubator. Determination of cell survival Chemotherapy survival was performed by the sulforhodamine B (SRB) assays. Cells (1!10 3 ) were plated in quadruplicate and cultured overnight. Drugs were added for 2 hours. Cells were incubated for 96 hours and stained with SRB for quantitation at 540 nm. Radiation survival was determined by the clonogenic ability by adding 500 cells in triplicate (0, 100, and 200 cgy), or 1000 cells (500 and 800 cgy). Colonies containing more than 50 cells were scored. When radiation was combined with chemotherapy, cells were pretreated with drugs for 4 (CDDP,

3 Saxena et al FU), or 16 hours (Tax). Ratio of surviving cells with radiation alone (500 cgy) was compared with combination of radiation and chemotherapy. Analysis of variance was used for statistical analysis. Radiation Cells were irradiated with 4 mv photons with a Phillips linear accelerator (Phillips Medical Systems, Andover, Mass) with an appositional beam at 100 cm SSD with 1 cm of tissue-equivalent lucite bolus in a field size of 39! 39 cm. Cells were positioned at central axis with a machine dose rate of approximately 400 cgy min ÿ1. Western blot analysis Cells were harvested in lysis buffer and samples (40 mg) were electrophoresed on 8% to 16% polyacrylamide gradient gels. Proteins were transferred to nitrocellulose, blocked with 5% nonfat dry milk, and probed with antibodies to mcl-1, survivin, AKT, pakt (Thr 308), pakt (Ser 473), p53, and p73. Peroxidase-labeled secondary antibody was used, and immunoreactive bands were visualized with electrochemoluminescent. Results Differences in response to the chemotherapeutic agents were observed in the 4 cell lines. CDDP and 5-FU sensitivity were similar in SiHa and CaSki as the most resistant cells, followed by ME180 cells. SiHa and CaSki were resistant to CDDP and 5-FU, followed by ME180. C33A cells were most sensitive to both CDDP and 5-FU (Figure 1, A and B). The most resistant cell line to Tax was ME180, followed by SiHa, C33A, and CaSki (Figure 1, C). The C33A cell line was the most sensitive to radiation with the other 3 cell lines displaying similar survival (Figure 2). There was 50% survival at 200 cgy with C33A cells being the most sensitive. The other cell lines had 50% survival at doses from 370 cgy to 420 cgy. For the chemoradiotherapy experiments, radiation was administered at 500 cgy, whereas chemotherapeutic agents (CDDP, Tax, and 5-FU) were given at an LD 30 concentration (Figure 3). The most cytotoxic chemoradiation regimen was CDDP and 5-FU in CaSki and SiHa cells, containing high-risk HPV. Tax was not effective in these 2 cell lines. ME180 cells demonstrated higher cell kill with combination than chemotherapy alone. Combination of drugs and radiation did not enhance the sensitivity of C33A cells. Expression of gene products AKT, the 2 forms of phosphorylated AKT, mcl-1, survivin, and p53 were studied. p53 expression was demonstrated in the HPVnegative C33A cells and ME180 cells, but was minimal in the other cell lines (data not shown). There was no Figure 2 Radiation survival curve of cervical cancer cells (C33A, CaSki, ME180, SiHa). Colony formation assay was used to determine survival. The assays were performed twice in triplicate. correlation with the expression of mcl-l, p73, and phosphorylated forms of AKT and sensitivity of these cell lines. Survivin expression correlated with the HPV- 16 and 18 expression in the CaSki and SiHa cells and the pattern of resistance to CDDP and 5-FU (Figure 4). Constitutive expression of AKT was the lowest in SiHa and CaSki cells with high-risk HPV sequences. Comment Radiotherapy is the treatment of choice for patients with locally advanced cervical carcinoma resulting in progression-free survival rates of 70%. 6 To increase local control rates, chemotherapeutic agents are being incorporated into the treatment protocols. We conducted this study to determine the effectiveness of the 3 chemotherapeutic agents that have been used in combination with radiation in cervical and other solid tumors. We demonstrate that HPV-16 or 18-containing cell lines were the most resistant to CDDP and 5-FU, whereas the cell line that was HPV negative (C33A) was the most sensitive. HPV-dependent inactivation of apoptotic regulators such as p53 might result in the cellular survival advantage provided by high-risk HPV that showed low expression in these cell lines, higher in HPV 39 (C) ME180 and HPV (ÿ) C33A. With combined treatments, CDDP and 5-FU were effective in enhancing the cytotoxic effect of both modalities in the HPV(C) CaSki, SiHa, and ME180 cells. Tax resulted in lessening of the radiation effect. Other investigators have demonstrated no radiosensitizing effect of Tax with ZMK-1, CaSki, and MCF-7 cells. 7,8 HPV(ÿ) C33A cell survival was not affected by multimodality therapy but demonstrated significant sensitivity to each agent separately. The question of whether HPV ( ) tumors would be better served by sequential therapy to reduce toxicity needs to be studied.

4 1402 Saxena et al Figure 3 Effect of radiation and chemotherapeutic agents on cervical cancer cells (C33A, CaSki, ME180, SiHa). Radiation dose used in these experiments was 500 cgy alone or in combination with LD 30 dose of CDDP, 5-FU, or Tax. Colony formation assay was used to determine survival. Asterisks, significantly less surviving colonies from radiation alone, P!.05. Figure 4 Western immunoblot analysis of expression of survivin and AKT in cervical cancer cell lysates. Study of the markers of survival demonstrated that survivin expression correlated with drug resistance in CaSki and SiHa cells similar to a previous study in colorectal cells. 9 Survivin is a member of the inhibitors of apoptosis protein (IAP) family that is involved in control of cell division and inhibition of apoptosis. Survivin has been shown to be expressed in most human neoplasms and is associated with clinical progression. 10 Clinical data on survivin and AKT expression may be helpful in elucidation of the potential role of these gene products as markers of therapeutic response. Other groups have suggested that concomitant CDDP/radiotherapy regimens may result in higher level of local tumor control through additive toxicity and not radiosensitization. No correlation between the cellular sensitivities to CDDP and radiation were demonstrated. Our study suggests that the effect of CDDP or other chemotherapeutic agents is likely to be compromised in tumors bearing high-risk HPV, an observation that could have profound clinical implications. It appears that radiation partially reverses this resistance; however, other chemotherapeutic agents may afford greater cell kill. Additional studies with varying concentrations and

5 Saxena et al 1403 schedules of drugs and radiation would be helpful in understanding the effectiveness of chemoradiotherapy. References 1. Monsonego J, Bosch FX, Coursaget P, Cos JT, Franco E, Frazer I, et al. Cervical cancer control, priorities and new directions. Int J Cancer 2004;108: Britten RA, Perdue S, Opoku J, Craighead P. Paclitaxel is preferentially cytotoxic to human cervical tumor cells with low Raf-1 kinase activity: implications for paclitaxel-based chemoradiation regimens. Radiother Oncol 1998;48: Lawrence TS, Blackstock AW, McGinn C. The mechanism of action of radiosensi-tization of conventional chemotherapeutic agents. Semin Radiat Oncol 2003;13: Peltenburg LTC. Radiosensitivity of tumor cells, oncogenes and apoptosis. Q J Nucl Med 2000;44: Eastham AM, Atkinson J, West CML. Relationships between clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine human cervix carcinoma cell lines. Int J Radiat Biol 2001;77: Rose PG. Chemoradiotherapy for cervical cancer. Eur J Cancer 2002;38: Pradier O, Rave-Frank M, Lehmann J, Lucke E, Boghun O, Hess CF, et al. Effects of docetaxel in combination with radiation on human head and neck cancer cells (ZMK-1) and cervical squamous cell carcinoma cells (CASKI). Int J Cancer 2001;91: Pradier O, Rave-Frank M, Schmidberger H, Bomecke M, Lehmann J, Meden H, et al. Effects of paclitaxel in combination with radiation on human head and neck cancer cells (ZMK-1), cervical squamous cell carcinoma (CaSki), and breast adenocarcinoma cells (MCF-7). J Cancer Res Clin Oncol 1999;125: Kennedy AS, Harrison GH, Mansfield CM, Zhou XJ, Xu JF, Balcer-Kubiczek EK. Survival of colorectal cancer cell lines treated with paclitaxel, radiation, and 5-FU: effect of TP53 or hmlh1 Deficiency. Int J Cancer 2000;90: Zaffaroni N, Daidone MG. Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat 2002;5:65-72.