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It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53(-/-) xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53(-/-) and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53(-/-) tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

No abstract

Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.

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Purpose Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity. We investigated antitumor activity of alofanib (RPT835), a novel allosteric FGFR2 inhibitor, in ovarian cancer in vitro and in vivo. Methods Equal amounts of ovarian cancer cell (SKOV3) lysates were analyzed for FGFR1-3 protein expression using Wes. To assess the efficacy of alofanib on FGF-mediated cell proliferation, SKOV3 cells were incubated and were treated with serially diluted alofanib. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of SKOV3 cancer cells. Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3 days during 31 days after tumor inoculation. Number of tumor vessels and Ki-67 index were calculated. Results SKOV3 cells express FGFR1 and FGFR2 but not FGFR3. Basic FGF increased proliferation of the ovarian cancer cells in untreated control group (P = 0.001). Alofanib inhibited growth of FGFR2-expressing SKOV3 cells with GI50 value of 0.37 μmol/L. Treatment with alofanib in combination with paclitaxel/carboplatin resulted in tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Compound exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350 mg/kg) demonstrated significant effect (inhibiting growth by 80 % and by 53 % in comparison with vehicle and chemotherapy group alone, respectively (P < 0.001). Alofanib decreased number of vessels in tumor (-49 %; P < 0.0001) and number of Ki-67-positive SKOV3 cells (-42 %, P < 0.05). There were tumor necrosis and cell degeneration in alofanib group. Conclusions We suggest that FGFR2 inhibition has potent effects on ovarian cancer growth in preclinical studies.

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Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes.We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines.Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations.In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.