Cytotoxic Activity Of Gemcitabine Research Articles

Implication of methylselenocysteine in combination chemotherapy with gemcitabine for improved anticancer efficacy

The limitations associated with cancer monotherapy including dose dependent toxicity and drug resistance can be addressed by combination chemotherapy. The combination of antineoplastic agents improves the cytotoxic activity in comparison to the single-agent based therapy in a synergistic or an additive mode by reducing tumor growth as well as metastatic ability. In the present investigation, we explored the potential of methylselenocysteine (MSC) in combination chemotherapy with gemcitabine (GEM). The cytotoxic activity of GEM and MSC was determined in various cell lines and based on the activity, A549 cells were explored for the mechanistic studies including DAPI staining, measurement of oxidative stress, mitochondrial membrane potential loss, nitric oxide level, western blotting, cell migration and colony formation assays. A549 cells in combination treatment with MSC and GEM demonstrated enhanced cytotoxicity with more irregular cellular morphology as well as chromatin condensation and nuclear blebbing. The selected combination also significantly triggered ROS generation and mitochondrial destabilization, and alleviated cell migration potential and clonogenic propensity of A549 cells. Also, caspase-3 and PARP mediated apoptosis was observed in the combination treated cells. MSC based drug combination could offer the attributes of improved drug delivery and there was a 6-folds dose reduction of GEM in combination. Further, antitumor study in Ehrlich solid tumor model showed the efficacy of MSC combination with GEM for the enhanced antitumor activity. The proposed combination demonstrated the potential for further translational studies.

Discovery of a novel chimeric ubenimex–gemcitabine with potent oral antitumor activity

Herein, a novel mutual prodrug BC-A1 was discovered by integrating ubenimex and gemcitabine into one molecule. Biological characterization revealed that compound BC-A1 could maintain both the anti-CD13 activity of ubenimex and the cytotoxic activity of gemcitabine in vitro. Further characterization also demonstrated that compound BC-A1 exhibited significant anti-invasion and anti-angiogenesis effects in vitro. The preliminary stability test of BC-A1 revealed that it could release gemcitabine in vitro. The in vivo anti-tumor results in liver cancer showed that at the same dosage, oral administration of BC-A1 was as potent as intraperitoneal administration of gemcitabine. This warranted the further research and development of the orally active prodrug BC-A1 because gemcitabine can not be orally administrated in clinic.

MP61-18 ELK-1 PROMOTES PROSTATE CANCER PROGRESSION AND SILODOSIN THAT INACTIVATES ELK-1 IN PROSTATE CANCER CELLS INCREASES SENSITIVITY TO GEMCITABINE

INTRODUCTION AND OBJECTIVES: LK-1 is a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation. However, little is known about biological significance of ELK1 in prostate cancer. In addition, silodosin, a selective a1-adrenergic blocker used for the symptomatic treatment of benign prostatic hyperplasia, has been shown to decrease ELK-1 expression in human prostate smooth muscle cells. In this study, we aim to investigate the functions of ELK-1 in prostate cancer growth and the efficacy of silodosin, in combination with chemotherapeutic drugs, in cell proliferation. METHODS: We first immunohistochemically determined the levels of phospho-ELK-1 (pELK-1) expression in radical prostatectomy specimens. We then assessed the effects of ELK-1 knockdown via short hairpin RNA on cell proliferation, migration, and invasion as well as those of silodosin on ELK-1 expression/activity in prostate cancer lines and their viability. RESULTS: Immunohistochemistry showed that the levels of pELK-1 expression were significantly higher in carcinoma (positivity & score, P<0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (positivity & score, P<0.001) than in benign as well as in carcinoma than in HGPIN (positivity, P1⁄40.330; score, P1⁄40.002). Although none of the 7 patients with pELK-1-negative carcinoma had biochemical recurrence after radical prostatectomy, Kaplan-Meier and log-rank tests revealed no significant correlations of pELK-1 levels with patient outcomes. In PC3 and DU145 cells expressing ELK-1 (mRNA, protein), ELK-1 silencing resulted in decreases in the expression of cfos, a downstream target of ELK-1 signals, as well as in cell viability/ colony formation, cell migration/invasion, and the expression of matrix metalloproteinase (MMP)-2 and MMP-9. In these cells, silodosin reduced the expression of a1-adrenergic receptor, ELK-1, and c-fos as well as the transcriptional activity of ELK-1. However, silodosin alone (up to 10 mM) only marginally inhibited PC3/DU145 cell growth. Interestingly, silodosin treatment in the prostate cancer lines increased sensitivity to gemcitabine, but not to cisplatin or docetaxel. CONCLUSIONS: ELK-1 is likely activated in prostate cancer cells. Our results also suggest that ELK-1 plays an important role in prostate cancer progression. Thus, ELK-1 inhibition has the potential of being a therapeutic approach for prostate cancer. Furthermore, silodosin that inactivates ELK-1 in prostate cancer cells enhances the cytotoxic activity of gemcitabine.

MP61-19 A NOVEL STRATEGY TO OPTIMISE TREATMENT OF DOCETAXEL-RESISTANT PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: LK-1 is a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation. However, little is known about biological significance of ELK1 in prostate cancer. In addition, silodosin, a selective a1-adrenergic blocker used for the symptomatic treatment of benign prostatic hyperplasia, has been shown to decrease ELK-1 expression in human prostate smooth muscle cells. In this study, we aim to investigate the functions of ELK-1 in prostate cancer growth and the efficacy of silodosin, in combination with chemotherapeutic drugs, in cell proliferation. METHODS: We first immunohistochemically determined the levels of phospho-ELK-1 (pELK-1) expression in radical prostatectomy specimens. We then assessed the effects of ELK-1 knockdown via short hairpin RNA on cell proliferation, migration, and invasion as well as those of silodosin on ELK-1 expression/activity in prostate cancer lines and their viability. RESULTS: Immunohistochemistry showed that the levels of pELK-1 expression were significantly higher in carcinoma (positivity & score, P<0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (positivity & score, P<0.001) than in benign as well as in carcinoma than in HGPIN (positivity, P1⁄40.330; score, P1⁄40.002). Although none of the 7 patients with pELK-1-negative carcinoma had biochemical recurrence after radical prostatectomy, Kaplan-Meier and log-rank tests revealed no significant correlations of pELK-1 levels with patient outcomes. In PC3 and DU145 cells expressing ELK-1 (mRNA, protein), ELK-1 silencing resulted in decreases in the expression of cfos, a downstream target of ELK-1 signals, as well as in cell viability/ colony formation, cell migration/invasion, and the expression of matrix metalloproteinase (MMP)-2 and MMP-9. In these cells, silodosin reduced the expression of a1-adrenergic receptor, ELK-1, and c-fos as well as the transcriptional activity of ELK-1. However, silodosin alone (up to 10 mM) only marginally inhibited PC3/DU145 cell growth. Interestingly, silodosin treatment in the prostate cancer lines increased sensitivity to gemcitabine, but not to cisplatin or docetaxel. CONCLUSIONS: ELK-1 is likely activated in prostate cancer cells. Our results also suggest that ELK-1 plays an important role in prostate cancer progression. Thus, ELK-1 inhibition has the potential of being a therapeutic approach for prostate cancer. Furthermore, silodosin that inactivates ELK-1 in prostate cancer cells enhances the cytotoxic activity of gemcitabine.

Synthesis, characterization and mechanistic-insight into the anti-proliferative potential of PLGA-gemcitabine conjugate.

Gemcitabine, a nucleoside analogue, is used in the treatment of various solid tumors, however, its efficacy is limited by rapid metabolism by cytidine deaminase and fast kidney excretion. In this study, a polymeric conjugate of gemcitabine was prepared by covalent coupling with poly(lactic-co-glycolic) acid (PLGA), in order to improve anticancer efficacy of the drug. The prepared conjugate was characterized by various analytical techniques including FTIR, NMR and mass spectroscopic analysis. The stability study indicated that the polymeric conjugate was more stable in plasma as compared to native gemcitabine. Further, in vitro cytotoxicity determined in a panel of cell lines including pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116), indicated that the cytotoxic activity of gemcitabine was retained following conjugation with polymeric carrier. In the nucleoside transportation inhibition assay, it was found that the prepared conjugate was not dependent on nucleoside transporter for entering into the cells and this, in turn, reflecting potential implication of this conjugate in the therapy of transporter- deficient resistance cancer. Further, the cell cycle analysis showed that the sub-G1 (G0) apoptotic population was 46.6% and 60.6% for gemcitabine and PLGA gemcitabine conjugate, respectively. The conjugate produced remarkable decrease in mitochondrial membrane potential, a marker of apoptosis. In addition, there was a marked increase in PARP cleavage and P-H2AX expression with PLGA gemcitabine conjugate as compared to native gemcitabine indicating improved apoptotic activity. The findings demonstrated the potential of PLGA gemcitabine conjugate to improve clinical outcome of gemcitabine based chemotherapy of cancer.

Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines

We aimed at investigating in vitro the cytotoxic activity (determined using WST-1, apoptosis and cell cycle assays) of gemcitabine, alone or in combination with mitotane, in mitotane-sensitive H295R and mitotane-insensitive SW-13 cells. Results of these experiments were compared with drug-induced modulation of RRM1 gene, the specific target of gemcitabine. In H295R cells, mitotane and gemcitabine combinations showed antagonistic effects and interfered with the gemcitabine-mediated inhibition of the S phase of the cell cycle. By contrast, in SW-13 cells, except when mitotane was sequentially administered prior to gemcitabine, the combination of the two drugs was synergistic. Such opposite effects were associated with opposite expression profiles of the target gene, with significant up-modulation in H295R but not in SW-13 under gemcitabine and mitotane combination treatment.

Study of Apoptosis Induction and Deoxycytidine Kinase/Cytidine Deaminase Modulation in the Synergistic Interaction of a Novel Ceramide Analog and Gemcitabine in Pancreatic Cancer Cells

This study investigated the interaction between the novel ceramide analog AL6 and gemcitabine in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, harboring different polymorphic variants of the gemcitabine catabolism enzyme cytidine deaminase (CDA). AL6 dose-dependently inhibited cell growth, induced apoptosis and synergistically enhanced the cytotoxic activity of gemcitabine. Moreover, it triggered apoptosis, which was significantly enhanced by the combination, and increased the ratio between gene expression of the activating enzyme deoxycytidine kinase (dCK) and CDA, potentially favoring gemcitabine activity. In conclusion, AL6 displays synergistic cytotoxic activity, enhances apoptosis, and favorably modulates enzymes involved in gemcitabine metabolism, supporting future investigation of this combination in pancreatic cancer.

Cytotoxic activity of gemcitabine in cultured cell lines derived from histologically different types of bladder cancer: Role of thymidine kinase 2

World wide incidence of bladder cancer is rising with nearly 13,760 deaths attributed to bladder cancer in 2007 in the USA. Tumor types of the urothelium include transitional cell carcinomas, squamous cell carcinomas, and adenocarcinomas. This study was undertaken to determine gemcitabine's efficacy against bladder cancer cell lines of different origins (HTB2, a papilloma; HTB3, a squamous cell carcinoma; and HTB4, a transitional cell carcinoma). Roles of nucleoside transporters and key enzymes in gemcitabine pharmacology were examined on the premise that cells originating from different types of bladder cancer exhibit different levels and/or types of nucleoside transporters and enzymes and thus may respond differently to gemcitabine. HTB2 cells had the highest transport efficiency and were also most responsive to gemcitabine. HTB3 and HTB4 cells had similar transport efficiencies, but exhibited different sensitivities to gemcitabine (HTB4>HTB3). The highest accumulation of [3H]gemcitabine was in HTB2 cells and the lowest was in HTB3 cells. Sequencing experiments revealed no mutations either in coding exons or intron–exon boundaries of the hENT1 genes of the three cell lines. HTB3 cells exhibited high thymidine kinase 2 (TK2) activity whereas HTB2 and HTB4 cells lacked detectable TK2 activity and pretreatment of HTB3 but not of HTB2 and HTB4 cells with extracellular thymidine resulted in enhanced sensitivity to gemcitabine. Our results highlight the importance of hENT1 and TK2 activities in response to gemcitabine. Elevated TK expression in squamous cell carcinomas warrants further study and offers new insights into rational treatment strategies based on bladder cancer phenotype.

Cytotoxic activity of gemcitabine and correlation with expression profile of drug-related genes in human lymphoid cells

Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA polymerization with promising activity in hematologic malignancies. Gemcitabine enters the cell mostly via the human equilibrative nucleoside transporter-1 (hENT1), while drug metabolism occurs by phosphorylation by deoxycytidine kinase (dCK), 5′-nucleotidase (cN-II) and cytidine deaminase (CDA) are the main inactivating enzymes. The aim of this study was to investigate the role of these determinants in gemcitabine cytotoxicity and analyze their expression in lymphoid cells. Cytotoxicity was assessed by MTT, and modulated by simultaneous addition of 2′-deoxycytidine (dCK natural substrate), tetrahydrouridine (CDA competitive inhibitor) and diethylpyrocarbonate (cN-II non-competitive inhibitor), while the expression of hENT1, dCK, cN-II, CDA and RR in WIL2-S, Jurkat and CCRF-CEM cells as well as in lymphoid cells from 25 chronic lymphocytic B-leukemia (B-CLL) patients was studied with quantitative-PCR. Cell cycle modulation and induction of apoptosis were analyzed by cytofluorimetry and bisbenzimide staining. Gemcitabine was highly cytotoxic, increased the cells in S-phase and significantly enhanced apoptosis. The crucial role of metabolism in gemcitabine activity was confirmed by the significant modulation of cytotoxicity by inhibitors of dCK, CDA and cN-II. Furthermore, PCR demonstrated a correlation between gemcitabine sensitivity and expression of its determinants, and that their values were within those observed in patients. These data indicate that gemcitabine is cytotoxic against lymphoid cells, affecting cell cycle and apoptosis. Furthermore, chemosensitivity may be predicted on the basis of gene expression profile of critical determinants involved in gemcitabine mechanism of action, suggesting the use of pharmacogenetic profiling for treatment optimization.