A Multi-Mitochondrial Anticancer Agent that Selectively Kills Cancer Cells and Overcomes Drug Resistance.

Mitochondria are double-membrane-bound organelles involved mainly in supplying cellular energy, but also play roles in signaling, cell differentiation, and cell death. Mitochondria are implicated in carcinogenesis, and therefore dozens of lethal signal transduction pathways converge on these organelles. Accordingly, mitochondria provide an alternative target for cancer management. In this study, F16, a drug that targets mitochondria, and chlorambucil (CBL), which is indicated for the treatment of selected human neoplastic diseases, were covalently linked, resulting in the synthesis of a multi-mitochondrial anticancer agent, FCBL. FCBL can associate with human serum albumin (HSA) to form an HSA-FCBL nanodrug, which selectively recognizes cancer cells, but not normal cells. Systematic investigations show that FCBL partially accumulates in cancer cell mitochondria to depolarize mitochondrial membrane potential (MMP), increase reactive oxygen species (ROS), and attack mitochondrial DNA (mtDNA). With this synergistic effect on multiple mitochondrial components, the nanodrug can effectively kill cancer cells and overcome multiple drug resistance. Furthermore, based on its therapeutic window, HSA-FCBL exhibits clinically significant differential cytotoxicity between normal and malignant cells. Finally, while drug dosage and drug resistance typically limit first-line mono-chemotherapy, HSA-FCBL, with its ability to compromise mitochondrial membrane integrity and damage mtDNA, is expected to overcome those limitations to become an ideal candidate for the treatment of neoplastic disease.

Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma.

AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2 antisense RNA, and to explore its effects on liver cancer cell proliferation. METHODS: We studied the expression of COX-2 in 34 cases of hepatocellular carcinoma (HCC) and SMMC7402 and SMMC7721 by immunohistochemical technique. Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402 and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry. Cell proliferation was determined by colony-forming efficiency. RESULTS: We observed COX-2 expression in 82.4% of the HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06 x 10(12) PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G(1)/G(0) phase in SMMC7402 cell line. The difference of apoptotic index between the Ad-AShcox-2 group and control group was statistically significant (t( control group ) = 32.62 and t( Ad-LacZ ) = 10.93, P<0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7+/-0.94)% and (33.6+/-4.24)%, respectively. CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.

[The effects of adenovirus-mediated human COX-2 antisense RNA on the growth of hepatocellular carcinoma].

OBJECTIVE: To investigate the relationship between the expression of COX-2 and liver cancer and construct a recombinant adenovirus encoding human COX-2 antisense RNA, and then to investigate its effects on liver cancer cell proliferation. METHODS: The expression of COX-2 in 34 cases of hepatocellular carcinoma and in SMMC-7402 and SMMC-7721 cell lines was studied by using immunohistochemical techniques. The shuttle plasmid encoding anti-sense COX-2 was constructed by using cloning COX-2 cDNA fragment in the reverse direction into the pHCMVSPIA. Then the plasmid pJM17 and the shuttle plasmid were co-transferred into 293 cells with lipofectamine for homologous recombination to acquire recombinant adenovirus (Ad-AShcox-2), which was confirmed by PCR. Human hepatocellular carcinoma cell lines SMMC-7402 and SMMC-7721 were transduced in vitro. The cell apoptosis and cell cycle were analyzed by flow cytometry. The cell proliferation was determined by colony-forming efficiency. RESULTS: We observed COX-2 expression in 82.4% of the hepatocellular carcinomas and SMMC-7402 cell line, but no COX-2 expression in the SMMC-7721 cell line. In addition, the recombinant adenovirus encoding anti-sense COX-2 fragment Ad-AShcox-2 was obtained with a titer of 1.06 x 10(12) PFU/ml. Ad-AShcox-2 reduced the expression of COX-2 and enhanced the percentage of cells into G1/G0 phase in the SMMC-7402 cell line. The difference of apoptotic index between the Ad-AShcox-2 group and the control group was statistically significant in SMMC-7402 but not in SMMC-7721. Similarly, colony-forming rates of SMMC-7402 and SMMC-7721 cell lines after Ad-AShcox-2 being transferred were (2.7+/-0.94)% and (33.6+/-4.24)%, respectively. CONCLUSION: By reducing the expression of COX-2 in hepatocellular carcinoma cells with the expression of COX-2, the cells could be inhibited.

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenografts.

AIM: To investigate the growth suppression of adenovirus expressing p27(kip1) on established esophageal tumors in nude mice. METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed recombinant adenoviral vectors carrying p27(kip1) gene (Ad-p27(kip1)) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27(kip1) and survivin was detected in tumors by immunohistochemical technique. RESULTS: The growth of tumors in gene therapy group with Ad-p27(kip1) was obviously suppressed compared to control group (0.42+/- 0.08 g vs 1.17+/- 0.30 g, t=6.39, P< 0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of p27(kip1) was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27(kip1). CONCLUSION: p27(kip1) gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27(kip1) expression and down-regulated survivin expression may be its important mechanisms.

Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells.

AIM: To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells. METHODS: Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by (3)H-thymidine ((3)H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27(kip1) was detected by immunocytochemical technique. RESULTS: After exposed to MG-132, the growth and value of (3)H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 micromol/L of MG-132 for 24, 48, 72 and 96 h (P<0.01). The percentage of cells at G(0)/G(1) phase was increased and that at S and G(2)/M was decreased (P<0.01). The rate of apoptotic cells treated with 5 micromol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27(kip1) were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group. CONCLUSION: MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27(kip1) expression, G(1) arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.

Inhibition of adenovirus-mediated p27kip1 gene on growth of esophageal carcinoma cell strain.

AIM: To investigate the inhibition of p27kip1 gene on the growth of esophageal carcinoma cell strain (EC9706). METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effect on p27kip1 expression, the growth of esophageal carcinoma cell, DNA replication, protein synthesis, cell multiplication and apoptosis were explored by means of cell growth count, 3H-TdR, 3H-Leucine incorporation, flow cytometry, DNA fragment analysis and TUNEL. RESULTS: Recombinant adenovirus Ad-p27kip1 was successfully constructed with a virus titer of 1.24 X 10(12) pfu/ml. p27kip protein expression increased markedly after EC-9706 transfection, while incorporation quantity of 3H-TdR and 3H-Leucine decreased significantly. The growth of esophageal carcinoma cell was inhibited obviously. Testing of flow cytometry displayed a typical apoptosis peak, and DNA gel electrophoresis showed a typical apoptosis ladder. TUNEL showed the apoptosis rate of Ad-p27kip1 group and control group to be 37.3% and 1.26% (P<0.001) respectively. CONCLUSION: Ad-p27kip1 can inhibit the growth and multiplication of esophageal carcinoma cells and induce apoptosis. Therefore, enhanced p27kip1 expression may be a new way to treat esophageal carcinoma.