Chandresh Sharma

Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer

BackgroundMitochondrial DNA (mtDNA) is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals. Alterations in the non-coding displacement (D) loop of mitochondrial DNA are present in many cancers. It has been suggested that the extent of mitochondrial DNA mutations might be useful in the prognosis of cancer outcome and/or the response to certain therapies. In order to investigate whether a high incidence of mutations exist in mitochondrial DNA of cervical cancer patients, we examined the frequency of mutations in the D-loop region in 19 patients of cervical cancer.ResultsMutations, often multiple, were detected in 18 of 19 (95%) patients. The presence of mutations correlated with Human Papilloma Virus (HPV) infection in these patients. Mutations were also detected in normal samples and lymphocytes obtained from cervical cancer patients, but their frequency of occurrence was much lower as compared to the cervical cancer tissues.ConclusionOur findings indicate that D-loop alterations are frequent in cervical cancers and are possibly caused by HPV infection. There was no association of mtDNA D-loop mutations with the histopathological grade and tumor staging.

Induction of Mitochondrial-Mediated Apoptosis by Morinda Citrifolia (Noni) in Human Cervical Cancer Cells

Cervical cancer is the second most common cause of cancer in women and has a high mortality rate. Cisplatin, an antitumor agent, is generally used for its treatment. However, the administration of cisplatin is associated with side effects and intrinsic resistance. Morinda citrifolia (Noni), a natural plant product, has been shown to have anti-cancer properties. In this study, we used Noni, cisplatin, and the two in combination to study their cytotoxic and apoptosis-inducing effects in cervical cancer HeLa and SiHa cell lines. We demonstrate here, that Noni/Cisplatin by themselves and their combination were able to induce apoptosis in both these cell lines. Cisplatin showed slightly higher cell killing as compared to Noni and their combination showed additive effects. The observed apoptosis appeared to be mediated particularly through the up-regulation of p53 and pro-apoptotic Bax proteins, as well as down- regulation of the anti-apoptotic Bcl-2, Bcl-XL proteins and survivin. Augmentation in the activity of caspase-9 and -3 was also observed, suggesting the involvement of the intrinsic mitochondrial pathway of apoptosis for both Noni and Cisplatin in HeLa and SiHa cell lines.

Modulation of HIV peptide antigen specific cellular immune response by synthetic α- and β-defensin peptides

Defensin peptides have their direct role in host defense against microbial infection as innate molecules and also thought to contribute to adaptive immunity by recruiting naïve T-cells and immature dendritic cells at the site of infection through CCR6 receptor. The main aim of the present study is to investigate the efficacy of defensins for the induction of cell mediated immune response against the peptide antigen of HIV-1 encapsulated in PLG microparticles through intranasal (IN) route in mice model. To characterized, we have analyzed T-cell proliferation, Th1/Th2 cytokines, β-chemokines production and IFN-γ/perforin secretion from CD4(+)/CD8(+) T-cells in response to HIV immunogen alone and with defensins at different mucosal site i.e. lamina propria (LP), spleen (SP) and peyers patches (PP). The cellular immunogenicity of HIV peptide with defensin formulations showed a significantly higher (p<0.001) proliferation response as compared to individual HIV peptide. The enhanced cytokines measurement profile showed mixed Th1 and Th2 type of peptide specific immune response by the incorporation of defensins. In the continuation, enhancement in MIP-1α and RANTES level was also observed in HIV peptide-defensin formulations. The FACS data had revealed that CD4(+)/CD8(+) T-cells showed significantly (p<0.001) higher IFN-γ and perforin secretion in HIV with defensin peptide formulations than HIV antigen alone group. Thus, the study emphasized here that defensin peptides have a potential role as mucosal adjuvant, might be responsible for the induction of cell mediated immunity when administered in mice through IN route with HIV peptide antigen.

Human papillomavirus 16 L1–E7 chimeric virus like particles show prophylactic and therapeutic efficacy in murine model of cervical cancer

Cervical cancer is found to be associated with human papillomavirus (HPV) infection, with HPV16 being the most prevalent. An effective vaccine against HPV can thus, be instrumental in controlling cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infection as well as cell-mediated immunity to eliminate established infection. In this study, we have generated a potential preventive and therapeutic candidate vaccine against HPV16. We expressed and purified recombinant HPV16 L1(ΔN26)-E7(ΔC38) protein in E. coli which was assembled into chimeric virus like particles (CVLPs) in vitro. These CVLPs were able to induce neutralizing antibodies and trigger cell-mediated immune response, in murine model of cervical cancer, exhibiting antitumor efficacy. Hence, this study has aimed to provide a vaccine candidate possessing both, prophylactic and therapeutic efficacy against HPV16 associated cervical cancer.

A synthetic chimeric peptide harboring human papillomavirus 16 cytotoxic T lymphocyte epitopes shows therapeutic potential in a murine model of cervical cancer

Abstract Infection with human papillomavirus (HPV) such as HPV16 is known to be associated with cervical cancer. The E6 and E7 oncoproteins of this virus are attractive targets for T-cell-based immunotherapy to cervical cancer. In our study, software predicted, multiple H-2Db restricted HPV16 cytotoxic T lymphocytes (CTL) epitopes on a synthetic chimeric peptide, was used along with different immunopotentiating adjuvants such as alum, heat-killed Mycobacterium w (Mw) cells, and poly d,l-lactic-co-glycolide (PLGA) microspheres. We have shown that subcutaneous immunization with H-2Db-restricted HPV16 peptide was able to generate CTL-mediated cytolysis of HPV16 E6- and E7-expressing TC-1 tumor cells in vitro, as well as protect against in vivo challenge with TC-1 cells in C57BL/6 mice. In vitro, this chimeric peptide showed best efficacy with PLGA microspheres, moderate with alum, and least with Mw as adjuvant. This approach may thus provide a potential peptide-based therapeutic candidate vaccine for the control of HPV infection and hence cervical cancer.

Response of primary culture of human ovarian cancer cells to chemotherapy: In vitro individualized therapy.

OBJECTIVE This study focused on whether primary cultures of ovarian cancer (OC) cells established from ascites can be used to evaluate response to chemotherapeutic agents and if curcumin could enhance the efficacy of these agents. MATERIALS AND METHODS We established five primary cultures of ascitic cells from OC patients and treated them with curcumin, carboplatin, and paclitaxel singly and in combinations. The percentage of apoptotic cells was determined by flow cytometry. RESULTS There was a wide variation in the response of individual primary cultures to treatment with the chemotherapeutic agents. Curcumin by itself was as good as carboplatin or paclitaxel in inducing apoptosis in the primary OC cells. Curcumin was not able to affect the carboplatin mediated cell killing. However, a combination of curcumin and paclitaxel was additive and was equally effective as a combination of carboplatin and paclitaxel. A combination of curcumin carboplatin, and paclitaxel was also found to be additive and, in fact, turned out to be the best combination that gave the highest percentage of apoptosis in vitro. CONCLUSION This study highlights the fact that primary cultures of OC cells can be used to detect response to chemotherapeutic agents and help to individualize the treatment offered to OC patients.

Development of chimeric candidate vaccine against HPV18: a proof of concept

AbstractHuman papillomaviruses (HPVs) are prerequisite for the development of cervical cancer, with HPV16 and HPV18 being the most prevalent. Despite the fact that two prophylactic vaccines against HPVs are in the market, wide-scale application of the vaccine in developing countries is a major problem as far as cost of the vaccine and lack of therapeutic efficacy are concerned. Hence, the aim of our study was to develop HPV18 L1E7 chimeric virus-like particles (CVLPs) vaccine candidate possessing both, prophylactic and therapeutic potential against HPV18-associated cervical cancer. In this study, we have developed a potential candidate vaccine against HPV18 involving HPV18 L1E7 CVLPs, which was expressed in E. coli and assembled in vitro. These CVLPs were able to induce a neutralizing antibody response as well as a cell-mediated immune response in mice.

Abstract 219: Treatment with survivin siRNA augments cytotoxicity of paclitaxel in primary ovarian cancer cells

Introduction Chemoresistance is a major problem encountered during the treatment of ovarian cancer. Paclitaxel one of the first line drugs is less effective in patients whose cancer cells overexpress survivin. This study investigates whether reducing expression of survivin by siRNA treatment of primary cultures of ovarian cancer cells can increase sensitivity to paclitaxel. Methodology Five primary cultures were established from ascites obtained from untreated ovarian cancer patients. Ascites mixed with MCDB 105 and M199 medium (ratio 1:1) was plated in 75 mm 2 flasks for establishment of primary cultures. Survivin siRNA was synthesized using in-vitro transcription technique and was quantified using pico green. A dose of 15 nM of Survivin siRNA was used for further experiments. Primary cultures were treated with survivin siRNA using olgofectamine as the transfecting agent. After 24 hours Survivin expression was quantified by RT-PCR. Cells plated in 96 well plates were treated with survivin siRNA (15nM). After 24 hours, paclitaxel treatment was given at four different doses (0.1, 1, 5 and 25 µg/ml) in separate wells. Untransfected cells, cells transfected with oligofectamine alone (mock transfection) and with universal control siRNA were also treated with same doses of paclitaxel after 24 hours. Cell survival was quantified using MTT assay. All experiments were done in triplicates. Results On treating the primary cultures with survivin siRNA there was a 6 fold decrease in Survivin expression. Survivin siRNA alone caused 23 ±3% cell deaths in the 5 primary cultures. When these cultures were treated with paclitaxel alone (untansfected cells) at different doses (0.1, 1, 5 and 25 µg/ml) the average cell death was 21.2%, 22.4%, 26% and 37.5% respectively. There was no significant difference in cell death in mock transfected and control siRNA transfected cells when compared to untransfected cells. However when the cells were pretreated with survivin siRNA and then followed by same doses of paclitaxel after 24 hours, cell death increased to 52.8%, 55.4%, 60% and 70% respectively. All the primary cultures were resistant to paclitaxel treatment as 62.5% of the cells survived the highest dose of paclitaxel. Survivin siRNA treatment could significantly increase the sensitivity of the primary ovarian cancer cell cultures to paclitaxel. Conclusion Survivin appears to play a major role in the response of ovarian cancer cells to paclitaxel treatment and knocking off survivin expression may prove beneficial in the treatment of ovarian cancer patients with paclitaxel whose tumors overexpress Survivin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 219.