Pharmacological development of target-specific delocalized lipophilic cation-functionalized carboranes for cancer therapy

PURPOSE: Tumor cell heterogeneity and microenvironment represent major hindering factors in the clinical setting toward achieving the desired selectivity and specificity to malignant tissues for molecularly targeted cancer therapeutics. In this study, the cellular and molecular evaluation of several delocalized lipophilic cation (DLC)-functionalized carborane compounds as innovative anticancer agents is presented. METHODS: The anticancer potential assessment of the DLC-carboranes was performed in established normal (MRC-5, Vero), cancer (U-87 MG, HSC-3) and primary glioblastoma cancer stem (EGFRpos, EGFRneg) cultures. Moreover, the molecular mechanism of action underlying their pharmacological response is also analyzed. RESULTS: The pharmacological anticancer profile of DLC-functionalized carboranes is characterized by: a) a marked in vitro selectivity, due to lower concentration range needed (ca. 10 fold) to exert their cell growth-arrest effect on U-87 MG and HSC-3, as compared with that on MRC-5 and Vero; b) a similar selective growth inhibition behavior towards EGFRpos and EGFRneg cultures (>10 fold difference in potency) without, however, the activation of apoptosis in cultures; c) notably, in marked contrast to cancer cells, normal cells are capable of recapitulating their full proliferation potential following exposure to DLC-carboranes; and, d) such pharmacological effects of DLC-carboranes has been unveiled to be elicited at the molecular level through activation of the p53/p21 axis. CONCLUSIONS: Overall, the data presented in this work indicates the potential of the DLC-functionalized carboranes to act as new selective anticancer therapeutics that may be used autonomously or in therapies involving radiation with thermal neutrons. Importantly, such bifunctional capacity may be beneficial in cancer therapy

Th17 Cells Are Involved in the Local Control of Tumor Progression in Primary Intraocular Lymphoma

BACKGROUND: Th17 cells play an important role in the pathogenesis of many autoimmune diseases, but despite some reports of their antitumor properties, too little is known about their presence and role in cancers. Specifically, knowledge is sparse about the relation of Th17 to lymphoma microenvironments and, more particularly, to the microenvironment of primary intraocular B-cell lymphoma (PIOL), an aggressive lymphoma with a poor prognosis. METHODS AND PRINCIPAL FINDINGS: In this work, we investigated the presence of Th17 cells and their related cytokines in a syngeneic model of PIOL, a subtype of non-Hodgkin lymphoma. The very small number of lymphocytes trafficking in normal eyes, which represent a low background as compared to tumor-bearing eyes, allows us to develop the present model to characterize the different lymphocyte subsets present when a tumor is developing. IL-21 mRNA was expressed concomitantly with IL-17 mRNA in tumor-bearing eyes and intracellular expression of IL-17A and IL-21 in infiltrating CD4(+) T lymphocytes. Interestingly, IL-17A production by T cells was negatively correlated with tumor burden. We also showed that IL-21 but not IL-17 inhibits tumor cell proliferation in vitro. CONCLUSIONS: These data demonstrate that IL-17A and IL-21-producing CD4(+) T cells, referred as Th17 cells, infiltrate this tumor locally and suggest that Th17-related cytokines may counteract tumor progression via IL-21 production. Thus, Th17 cells or their related cytokines could be considered to be a new therapeutic approach for non-Hodgkin B-cell lymphomas, particularly those with an ocular localization

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

Natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants

Nuclear ING2 expression is reduced in human cutaneous melanomas

Cutaneous malignant melanoma is a severe and sometimes life-threatening cancer. The molecular mechanism of melanomagenesis is incompletely understood. Deregulation of apoptosis is probably one of the key factors contributing to the progression of melanoma. The inhibitor of growth (ING) family proteins are candidate tumour suppressors which play important roles in apoptosis. Downregulated expression of ING proteins have been reported in several tumour types, including the loss of nuclear expression of p33ING1b in melanoma. As ING2 exhibits 58.9% homology with p33ING1b, we hypothesized that the aberrant expression of ING2 may be involved in melanomagenesis. Here, we used tissue microarray technology and immunohistochemistry to examine ING2 expression in human nevi and melanoma biopsies. Our data showed that nuclear ING2 expression was significantly reduced in radial growth phase (RGP), vertical growth phase (VGP), and metastatic melanomas compared with dysplastic nevi (P<0.05). Our data also revealed that nuclear ING2 expression was not associated with patient's gender, age or tumour thickness, ulceration, American Joint Committee on Cancer (AJCC) stage, tumour subtype, location and 5-year survival (P>0.05). Taken together, our results suggest that nuclear ING2 expression is significantly reduced in human melanomas and that reduced ING2 may be an important molecular event in the initiation of melanoma development

Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Withaferin A (WA), a naturally occurring withanolide, induces apoptosis in both estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 breast cancer cell lines with higher sensitivity in MCF-7 cells, but the underlying mechanisms are not well defined. The purpose of this study was to determine the anti-cancer effects of WA in MCF-7 breast cancer cells and explore alterations in estrogen receptor alpha (ERα) and its associated molecules <it>in vitro </it>as novel mechanisms of WA action.</p> <p>Methods</p> <p>The effects of WA on MCF-7 viability and proliferation were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and trypan blue exclusion assays. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle effects were analyzed by PI flow cytometry. Western blotting was also conducted to examine alterations in the expression of ERα and pathways that are associated with ERα function.</p> <p>Results</p> <p>WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h. It also caused a dose- and time-dependent apoptosis and G2/M cell cycle arrest. WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression. Co-treatment with protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132 revealed that depletion of ERα by WA is post-translational, due to proteasome-dependent ERα degradation.</p> <p>Conclusions</p> <p>Taken together, down-regulation of ERα, RET, HSF1 and up-regulation of phospho-p38 MAPK, p53, p21 are involved in the pro-apoptotic and growth-inhibitory effects of WA in MCF-7 breast cancer cells <it>in vitro</it>. Down-regulation of ERα protein levels by WA is caused by proteasome-dependent ERα degradation.</p

Reduced Apaf-1 expression in human cutaneous melanomas

Malignant melanoma is a life-threatening skin cancer due to its highly metastatic character and resistance to radio- and chemotherapy. It is believed that the ability to evade apoptosis is the key mechanism for the rapid growth of cancer cells. However, the exact mechanism for failure in the apoptotic pathway in melanoma cells is unclear. p53, the most frequently mutated tumour suppressor gene in human cancers, is a key apoptosis inducer. However, p53 mutation is only found in 15–20% of melanoma biopsies. Recently, it was found that Apaf-1, a downstream target of p53, is inactivated in metastatic melanoma. Specifically, loss of heterozygosity (LOH) of the Apaf-1 gene was found in 40% of metastatic melanoma. To determine if loss of Apaf-1 expression is indeed involved in melanoma progression, we employed the tissue microarray technology and examined Apaf-1 expression in 70 human primary malignant melanoma biopsies by immunohistochemistry. Our data showed that Apaf-1 expression is significantly reduced in melanoma cells compared with normal nevi (χ2=6.02, P=0.014). Our results also revealed that loss of Apaf-1 was not associated with the tumour thickness, ulceration or subtype, patient's gender, age and 5-year survival. In addition, our in vitro apoptosis assay revealed that overexpression of Apaf-1 can sensitise melanoma cells to anticancer drug treatment. Taken together, our data indicate that Apaf-1 expression is significantly reduced in human melanoma and that Apaf-1 may serve as a therapeutic target in melanoma

Polyphenols Sensitization Potentiates Susceptibility of MCF-7 and MDA MB-231 Cells to Centchroman

Polyphenols as “sensitizers” together with cytotoxic drugs as “inducers” cooperate to trigger apoptosis in various cancer cells. Hence, their combination having similar mode of mechanism may be a novel approach to enhance the efficacy of inducers. Additionally, this will also enable to achieve the physiological concentrations facilitating significant increase in the activity at concentrations which the compound can individually provide. Here we propose that polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC, antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in polyphenols alone. We conclude that differential sensitization of HBCCs with low dose polyphenol augments apoptotic efficacy of CC. This may offer a novel approach to achieve enhanced action of CC with concomitant reduction of side effects enabling improved management of hormone-dependent breast cancer