modulation of antitumor immune response in mouse

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Tbilisis saxelmwifo samedicino universiteti, alergologiisa da klinikuri imunologiis departamenti, saqarTvelo; 3virjiniis. Tanamegobrobis universiteti, urologiis ...

GEORGIAN MEDICAL NEWS No 5 (266) 2017

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MODULATION OF ANTITUMOR IMMUNE RESPONSE IN MOUSE PROSTATE CANCER MODEL 1

Mitskevich N., 1Tsertsvadze T., 1Mchedlishvili K., 2Matchavariani K., 3Guruli G.

Iv. Javakhishvili Tbilisi State University, Division of Immunology and Microbiology; Tbilisi State Medical University, Department of Allergology and Clinical Immunology, Georgia; 3 Virginia Commonwealth University, Division of Urology, Richmond VA, USA. 1

2

Prostate cancer is the fifth most common cancer in both sexes combined and the second most common cancer in men. It is also the most common cancer in American men, and second leading cause of cancer death among them., with more than 161 000 new cases expected in the United States in 2017 (American Cancer Society, 2017). In Georgia, 222 men were diagnosed with prostate cancer in 2008 (6th most common cancer in Georgian men), and 143 died with this disease (7th leading cause of cancer death). Obviously, this is a very important health problem worldwide. Though the treatment of the localized prostate cancer is well established and mostly successful, once disease spreads outside of prostate it becomes virtually incurable. The only effective treatment of the prostate cancer is hormonal therapy, which halts the progression of the disease, but does not eliminate it. In addition, basic principles of hormonal therapy (androgen ablation), which was the first successful therapy against advanced cancer, has not been changed much since its introduction more than 50 years ago. Available treatments for the advanced prostate cancer (secondary hormonal manipulations, chemotherapy, etc.) have only limited effectiveness and significant side effects. Therefore, search and development of the effective therapies for the advanced hormone-refractory prostate cancer is one of the critical problems of contemporary cancer treatment. Immunomodulatory drugs are being used with increasing frequency to treat various cancers. And although some of the greatest advancements have been achieved in the treatment of hematologic malignancies, a growing body of evidence exists to suggest a benefit in the treatment of solid organ tumors, including prostate cancer. Though the © GMN

benefits of therapy have been shown in clinical trials, the underlying immune mechanisms responsible for the outcomes have not been completely elucidated. With clinical research confirming the benefits of combination therapy over monotherapy, it has become more important than ever to define the effects each drug has upon the immune system and the target malignancy so that new regimens might be created to maximize efficacy and minimize side effects. As our understanding of tumor immunology has evolved, the role for immunotherapy in the treatment of malignancy has evolved as well. The discovery of cancer testis antigens (CTA) has fueled further research into both adaptive and adoptive immunotherapy techniques. Cancer testis antigens are expressed in the “immunologically privileged” tissues, and therefore autologous T cells do not have inherent tolerance toward them. With limited expression in adult tissue and increased expression in certain malignancies, cancer testis antigens seem ideal targets for immunotherapy. CTA expression is controlled epigenetically by CpG methylation in gene promoters and histone acetylation in neighboring chromatin. Therefore, CTA expression in tumor cells can be augmented by the administration of DNA methylation inhibitors [1-3]. Immunotherapy provides promise for the treatment of prostate cancer and other genitourinary (GU) malignancies. Early studies combining immunomodulatory drugs and chemotherapeutic agents have shown some benefit and prompted further investigation into their use as part of the medical armamentarium against prostate cancer. Current research focuses on two compounds currently being utilized in combination to treat hematologic malignancies, 39

МЕДИЦИНСКИЕ НОВОСТИ ГРУЗИИ CFMFHSDTKJC CFVTLBWBYJ CBF[KTYB

5-Azacytidine (5-AzaC) and Lenalidomide (Lena). Both, Lena and 5-AzaC have been used in combination with other chemotherapeutic agents with variable results in the treatment of prostate cancer. Current research focuses on two compounds currently being utilized in combination to treat hematologic malignancies, 5-Azacytidine (5-AzaC) and Lenalidomide. 5-AzaC and its analogues, DNA methylation inhibitors, have been shown to increase tumor exposure to the immune system through various mechanisms including upregulation of gene expression of cancer testis antigens, as well as through direct cytotoxic effects on tumor cells as a result of DNA demethylation [2-4]. It has also shown to have some effect on the immune system, though the exact mechanism of these effects not well known at this time [5-8]. Lenalidomide and its analogues, synthetic compounds created from thalidomide to be more potent with less neurologic side effects, have been shown to augment both innate and adaptive immune responses resulting in improved response to malignancies when combined with chemotherapeutic drugs [9]. It has also been confirmed that the drugs exhibit direct inhibitory effects on angiogenesis and tumor cell proliferation [9-12].Though a lot of research has been done on the effects these drugs have upon NK cells and T lymphocytes [13-16]. Not much is known about their effect on dendritic cells, and no studies to date have looked at their combination in the treatment of prostate cancer.

vivarium at Iv. Javakhishvili Tbilisi State University according to standard guidelines. Prostate Cancer Cell Line: RM-1 - a murine prostate cancer cell line and Study Drugs: Lenalidomide and 5-AzaCcitidine were gifted by Virginia Commonwealth University. Dendritic cells: briefly, bone marrow has been collected from tibias and femurs of Male C57BL/6 animals. At first, we evaluated the compounds (Lenalidomide, 5-Azacitidine) separately in vitro and studied their effects on prostate cancer RM-1 cells and dendritic cells (DC). We studied a proliferation of the cells using the Cell Proliferation Assay. Dendritic cells and RM-1 cells in suspension were exposed to different concentrations of lenalidomide. A DMSO control and one group with no treatment were also included. Flow cytometry was performed on dendritic cells after their stimulation with different concentrations of lenalidomide DC were stained with following antibodies: MHC class I, MHC class II, CD40, CD80, CD86, CD205. RT-PCR was performed on all groups looking at P1A, ETA and ETB gene expression. Expression of both endothelin genes was identified in all groups prompting performance of real time PCR

Material and methods. Mice: C57BL/6 and balb/c mice have been obtained. Animals were maintained at the

The production of IL-12 and IL-15 was identified in all groups using ELISA and their levels in the supernatants were determined.

Fig. 1. Dendritic cells proliferation assay. DC without stimulation and DC treated with DMSO (solvent for lenalidomide) provided controls. Lenalidomide was used at the concentration of 0.5µM, 1.0µM, 2.0µM and 10µM. No statistical difference was seen when comparing different conditions. DC – dendritic cells, LN - lenalidomide

Fig. 2. RM-1 prostate cancer cells proliferation assay. Untreated RM-1 cells and RM-1 cells treated with DMSO (solvent for lenalidomide) provided controls. Lenalidomide was used at the concentration of 0.5µM, 1.0µM, 2.0µM and 10µM. No statistical difference was seen in cell proliferation when comparing different conditions. LN - lenalidomide

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GEORGIAN MEDICAL NEWS No 5 (266) 2017

Results and their discussion. Cell proliferation Assay (Lenalidomide): Dendritic cells and RM-1 cells in suspension were exposed to 4 different concentrations of lenalidomide from 0.5- 10 μM. A DMSO control and one group with no treatment were also included. The cell proliferation assay was then performed, as described above, on each group. Dendritic cell proliferation increased slightly with increasing concentrations of lenalidomide. RM-1 cell proliferation increased up to concentrations of 2 μM after which it decreased in a dose dependent fashion. The differences in cell proliferation between groups did not reach statistical significance with either dendritic cells or RM-1 cells (Figs. 1 and 2). Cell proliferation Assay (5-Azacitidine): Dendritic cells and RM-1 cells in suspension were exposed to two different concentrations of 5-AzaC, 0.5 μM and 1.0 μM. One group with no treatment was also included as a control. The cell proliferation assay was then performed, as described above, on each group. Cell proliferation in both DC and RM-1 cells decreased in a dose dependent fashion with increasing concentrations of 5-AzaC. For the DC statistically significant difference from control was seen only at the concentration of 1.0 µM (P=0.029). Results are shown in Fig. 3.

Fig. 4. RM-1 cells proliferation assay. RM-1 cells without treatment provided control. 5-Azacitidine (5-AzaC) was used at the concentrations of 0.5µM and 1.0µM. Statistically significant difference in cell proliferation from the control (decrease) was seen at both 5-AzaC concentrations. 5-AzaC – 5-Azacitidine, RM-1 – murine prostate cancer cells * Depicts statistically significant difference in comparison to RM-1 cells Flow Cytometry (Dendritic Cells and Lenalidomide): Flow cytometry was performed on dendritic cells after their stimulation with different concentrations of lenalidomide. Dendritic cells exposed to DMSO (solvent for lenalidomide) acted as control. Lenalidomide was added during the last 48 hours of culture, after which DC were harvested, stained with different antibodies (MHC class I, MHC class II, CD40, CD80, CD86 and CD205) and flow cytometry was performed. Results are presented in Fig. 5.

Fig. 3. Dendritic cells proliferation assay. DC without stimulation provided control. 5-Azacitidine (5-AzaC) was used at the concentrations of 0.5µM and 1.0µM. Statistically significant difference in cell proliferation from the control (decrease) was seen at the 5-AzaC concentration of 1.0 µM. 5-AzaC – 5-Azacitidine, DC – dendritic cells * Depicts statistically significant difference in comparison to untreated DC For the RM-1 cells (Fig. 4) the difference between the control and both 5-AzaC treated cells were statistically significant (P=0.016 for the group treated with the concentration of 0.5 µM and P=0.004 for the group treated with the concentration of 1.0 µM of 5-AzaC). There was no statistically significant difference between the 5-AzaC treated groups. © GMN

Fig. 5. Flow cytometry results for DC treated with lenalidomide. DC treated with DMSO (0.1%, solvent for lenalidomide) provided control. Lenalidomide was used at the concentration of 0.5µM, 1.0µM, and 10µM. DMSO and lenalidomide were applied for the last 48 hours of culture. DC were stained with following antibodies: MHC class I, MHC class II, CD40, CD80, CD86, CD205. LN – lenalidomide, DC – dendritic cells * Depicts statistically significant difference in comparison to DMSO-treated DC 41

МЕДИЦИНСКИЕ НОВОСТИ ГРУЗИИ CFMFHSDTKJC CFVTLBWBYJ CBF[KTYB

As it can be seen, there was approximately 10% rise in the expression of most DC markers at the lenalidomide concentration of 0.5 µM, and these differences were statistically significant (P

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