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The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.

Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to >100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair. J. Cell. Physiol. 229: 1548–1556, 2014. © 2014 Wiley Periodicals, Inc.

Lung cancer is the leading cause of cancer mortality worldwide and despite efforts made to improve clinical results, continuing poor survival rates indicate that novel therapeutic approaches are needed. Valproic acid (VPA), a short-chain branched fatty acid used mainly for the treatment of epilepsy and bipolar disorder, has been shown to inhibit class I histone deacetylases (HDAC-I), a group of enzymes involved in chromatin remodeling and which are thought to play a role in tumor development. Although evidence of VPA's therapeutic efficacy has also been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical usefulness. We used a panel of NSCLC cell lines to evaluate the activity and mechanisms of action of organosulfur valproic acid derivatives, a promising new class of compounds designed to improve the safety and efficacy of the valproic acid molecule and created by coupling it with a hydrogen sulfide (H(2) S)-releasing moiety. Our results highlighted the increased cytotoxic activity of the novel organosulfur derivatives, ACS33 and ACS2, with respect to VPA, starting from low concentrations. In particular, ACS2 exhibited important pro-apoptotic activity triggered by the mitochondrial pathway and also showed anti-invasion potential. Furthermore, our in vitro results identified a highly effective combination schedule of ACS2 and cisplatin capable of inducing a synergistic interaction even when the two drugs were used at low concentrations, which could prove a valid alternative to traditional chemotherapeutic regimens used for advanced lung cancer. Further studies are needed to confirm these preliminary findings.

Sorafenib is a multikinase inhibitor that has shown promising therapeutic results in different tumor histotypes, both as a single agent or in combination with other treatments. We analyzed the in vitro activity of sorafenib in pancreatic cancer, one of the most lethal and chemo-radio-resistant tumors, using four human pancreatic cancer cell lines (t3m4, Capan 1, Capan 2, and MiaPaca 2), characterized by different K-ras gene status and RAF/MEK/ERK profile. Sorafenib exerted a strong anti-proliferative effect independently of RAS/RAF/MEK/ERK and induced various degrees of apoptosis in the cell lines. The mechanisms involved were explored in detail in t3m4 and Capan 1, in which sorafenib induced the highest and lowest levels of apoptosis, respectively. In t3m4, the RAF/AKT/STAT-3 rather than the RAF/MEK/ERK pathway was involved, whereas in Capan 1 cells there was a strong decrease in pMEK and pERK which was not accompanied by an important reduction in RAF, AKT, and STAT-3 proteins or in their phosphorylation. Moreover, U0126-induced MEK inhibition did not induce apoptosis in any cell line, reinforcing the hypothesis of a MEK/ERK-independent mechanism of sorafenib activity. Mcl-1 appears to play a crucial role in sorafenib-induced apoptosis. In fact, both protein and mRNA were downregulated in t3m4 and upregulated in Capan 1, in which siRNA-induced silencing resulted in the same level of apoptosis as observed in t3m4. Our results show that sorafenib exerts anti-proliferative and pro-apoptotic activity in pancreatic cancer cells. Used singly or in combination with other drugs, it could therefore represent valid treatment for pancreatic cancer.

Studies performed in different experimental and clinical settings have shown that Docetaxel (Doc) is effective in a wide range of tumors and that it exerts its activity through multiple mechanisms of action. However, the sequence of events induced by Doc which leads to cell death is still not fully understood. Moreover, it is not completely clear how Doc induces mitotic catastrophe and whether this process is an end event or followed by apoptosis or necrosis. We investigated the mechanisms by which Doc triggers cell death in hormone-refractory prostate cancer cells by analyzing cell cycle perturbations, apoptosis-related marker expression, and morphologic cell alterations. Doc induced a transient increase in G2/M phase followed by the appearance of G0/1 hypo- and hyperdiploid cells and increased p21 expression. Time- and concentration-dependent apoptosis was induced in up to 70% of cells, in concomitance with Bcl-2 phosphorylation, which was followed by caspase-2 and -3 activation. In conclusion, Doc would seem to trigger apoptosis in hormone-refractory prostate cancer cells via mitotic catastrophe through two forms of mitotic exit, in concomitance with increased p21 expression and caspase-2 activation.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the etiopathogenesis of lung cancer and is a promising tool for cancer detection. In the present study, promoter hypermethylation of p16 I N K 4 A and CDH 13 genes was investigated in tumor tissue and in matched serum from 61 patients with histologically confirmed non-small cell lung cancer. Using a fluorescence-based method of methylation-specific PCR(F-MSP), methylation of p16 I N K 4 A and CDH13was detected in 79% and 66% of tumors, respectively, and was not significantly related to conventional clinicopathological characteristics of patients or tumors. Methylation of both genes was observed in 52% of tumors and of at least one gene in 92% of lesions. In matched serum, hypermethylation of p16 I N K 4 A and CDH 13 was observed in 26% and 23% of patients, respectively, but as they were not associated, the methylation of at least one gene was detected in 39% of patients. In conclusion, the frequency of p16 I N K 4 A or CDH13 hypermethylation in patient serum, together with evidence of their early occurrence in lung cancerogenesis and the total lack of methylation in serum from healthy individuals, offer a promising tool for non invasive early detection of lung cancer.

Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation → cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair.

The efficacy of therapy for hormone-refractory prostate cancer (HRPC) is still unsatisfactory and new agents and therapeutic modalities are needed. The aims of the present work were to examine the in vitro activity and mechanisms of action of doxorubicin (DX), pegylated liposomal DX (PLDX), and non-pegylated liposomal DX (NPLDX) in DU145 and taxane-resistant DU145-R HRPC cell lines. Drug activity and incorporation, apoptosis, and expression of cell death-related markers were evaluated by SRB test, cytofluorimetric assays, and Western blot, respectively. Among the different DX formulations, NPLDX showed the highest cytotoxic activity in both cell lines, with more than 50% of apoptotic cells at only 1/10 of the plasma peak concentration after 72 h exposure. Anthracyclines, in particular NPLDX, were highly concentrated in the Golgi apparatus. Moreover, a significant increase was observed in the expression of CD95 receptor, GD3 ganglioside and, caspase-2 and -8 active forms in both cell lines followed by caspase-3 activation and mitochondrial membrane depolarization. The Golgi apparatus, probably acting as a stress sensor, intensified the conventional apoptotic mechanism induced by anthracyclines. Our data support the hypothesis that organelle-dependent initiation of cell death other than that induced by mitochondria and nucleus is a research area worthy of pursuing and suggest that the Golgi apparatus could be an ideal target for anti-cancer therapy. Of note, the activity of NLPDX in taxane-resistant DU145-R cells warrants further evaluation as second-line treatment of advanced HRPC after taxane failure. J. Cell. Physiol. 226: 3035–3042, 2011. © 2011 Wiley-Liss, Inc.