Nanocarrier - Mediated Salinomycin Delivery Induces Apoptosis and Alters EMT Phenomenon in Prostate Adenocarcinoma.

Salinomycin (Sal) has been recently discovered as a novel chemotherapeutic agent against various cancers including prostate cancer which is one of the most commonly diagnosed cancers affecting male populations worldwide. Herein we designed salinomycin nanocarrier (Sal-NPs) to extend its systemic circulation and to increase its anticancer potential. Prepared nanoform showed high encapsulation and sustained release profile for salinomycin. The present study elucidated the cytotoxicity and mechanism of apoptotic cell death of Sal-NPs against prostate cancer both in vitro and in vivo. At all measured concentrations, Sal-NPs showed more significant cytotoxicity to DU145 and PC3 cells than Sal alone. This effect was mediated by apoptosis, as confirmed by ROS generation, loss of MMP and cell cycle arrest at the G1 phase in both cells. Sal-NPs efficiently inhibited migration of PC3 and DU145 cells via effectively downregulating the epithelial mesenchymal transition. Also, the results confirmed that Sal-NPs can effectively inhibit the induction of Prostate adenocarcinoma in male Wistar rats. Sal-NPs treatment exhibited a decrease in tumour sizes, a reduction in prostate weight, and an increase in body weight, which suggests that Sal-NPs is more effective than salinomycin alone. Our results suggest that the molecular mechanism underlying the Sal-NPs anticancer effect may lead to the development of a potential therapeutic strategy for treating prostate adenocarcinoma.

Cooperative STAT3-NFkB signaling modulates mitochondrial dysfunction and metabolic profiling in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) continues to pose a significant health challenge and is often diagnosed at advanced stages. Metabolic reprogramming is a hallmark of many cancer types, including HCC and it involves alterations in various metabolic or nutrient-sensing pathways within liver cells to facilitate the rapid growth and progression of tumours. However, the role of STAT3-NFκB in metabolic reprogramming is still not clear. APPROACH AND RESULTS: Diethylnitrosamine (DEN) administered animals showed decreased body weight and elevated level of serum enzymes. Also, Transmission electron microscopy (TEM) analysis revealed ultrastructural alterations. Increased phosphorylated signal transducer and activator of transcription-3 (p-STAT3), phosphorylated nuclear factor kappa B (p-NFκß), dynamin related protein 1 (Drp-1) and alpha-fetoprotein (AFP) expression enhance the carcinogenicity as revealed in immunohistochemistry (IHC). The enzyme-linked immunosorbent assay (ELISA) concentration of IL-6 was found to be elevated in time dependent manner both in blood serum and liver tissue. Moreover, immunoblot analysis showed increased level of p-STAT3, p-NFκß and IL-6 stimulated the upregulation of mitophagy proteins such as Drp-1, Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Meanwhile, downregulation of Poly [ADP-ribose] polymerase 1 (PARP-1) and cleaved caspase 3 suppresses apoptosis and enhanced expression of AFP supports tumorigenesis. The mRNA level of STAT3 and Drp-1 was also found to be significantly increased. Furthermore, we performed high-field 800 MHz Nuclear Magnetic Resonance (NMR) based tissue and serum metabolomics analysis to identify metabolic signatures associated with the progression of liver cancer. The metabolomics findings revealed aberrant metabolic alterations in liver tissue and serum of 75th and 105th days of intervention groups in comparison to control, 15th and 45th days of intervention groups. Tissue metabolomics analysis revealed the accumulation of succinate in the liver tissue samples, whereas, serum metabolomics analysis revealed significantly decreased circulatory levels of ketone bodies (such as 3-hydroxybutyrate, acetate, acetone, etc.) and membrane metabolites suggesting activated ketolysis in advanced stages of liver cancer. CONCLUSION: STAT3-NFκß signaling axis has a significant role in mitochondrial dysfunction and metabolic alterations in the development of HCC.

Frequent expression of a novel cancer testis antigen, protein kinase human monopolar spindle 1 (hMps1/TTK) in human urinary bladder transitional cell carcinoma.

Urothelial bladder cancer (UBC) is a frequently occurring malignancy of the urinary tract. The present study was undertaken to evaluate the mRNA and immunohistochemical (IHC) expression of protein kinase human monopolar spindle 1 (hMps1/TTK) gene in transitional cell carcinoma (TCC) of the bladder and correlate its expression with the clinicopathological characteristics of patients. In the present study, quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate TTK mRNA expression in TCC. IHC analysis of TTK was also evaluated. Independent Student's t, ANOVA and chi-square (χ2) tests were used to analyze the data statistically. The frequency of TTK mRNA over expression was detected in 50% of UBC (38/76) by qRT-PCR. Relative mean fold expression of TTK mRNA was found significantly (p < 0.05) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (8.96 ± 4.51 vs. 5.64 ± 3.53, p = 0.03). Moreover, IHC reveals heterogenous immunostaining pattern of TTK in TCC tissues. The frequency of TTK protein over expression was detected in 56.9% (37 of 65) UBC patients. No significant IHC expression of TTK was detected among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Collectively our study observations conclude that TTK is a novel cancer/testis antigen (CTA) as a diagnostic marker for early diagnosis of UBC.

Piperine and Celecoxib synergistically inhibit colon cancer cell proliferation via modulating Wnt/ß-catenin signaling pathway.

BACKGROUND: Celecoxib (CXB), a selective COX-2 inhibitor NSAID, has exhibited prominent anti-proliferative potential against numerous cancers. However, its low bioavailability and long term exposure related cardiovascular side effects, limit its clinical application. In order to overcome these limitations, natural bioactive compounds with lower toxicity profile are used in combination with therapeutic drugs. Therfore, in this study Piperine (PIP), a natural chemo-preventive agent possessing drug bioavailability enhancing properties, was considered to be used in combination with low doses of CXB. PURPOSE: We hypothesized that the combination of PIP with CXB will have a synergistic anti-proliferative effect on colon cancer cells. STUDY DESIGN: The potency of PIP and CXB alone and in combination was evaluated in HT-29 human colon adenocarcinoma cells and mechanism of growth inhibition was investigated by analyzing the players in apoptotic and Wnt/ß-catenin signaling pathways. METHODS: The effect of PIP on the oral bioavailability of CXB in mice was investigated using HPLC analysis. The study investigated the synergistic anti-proliferative effect of CXB and PIP on HT-29 cells and IEC-6 non-tumorigenic rat intestinal epithelial cells by SRB cell viability assay. Further, the cellular and molecular mechanism(s) involved in the anti-proliferative combinatorial effect was extensively explored in HT-29 cells by flow cytometry and western blotting. The in vivo efficacy of this combination was studied in CT26.WT tumor syngeneic Balb/c mice model. RESULTS: PIP as a bioenhancer increased the oral bioavailability of CXB (129%). The IC50 of CXB and PIP were evaluated to select doses for combination treatment of HT-29 cells. The drug combinations having combination index (CI) less than 1 were screened using CompuSyn software. These combinations were significantly cytotoxic to HT-29 cells but IEC-6 were least effected. Further, the mechanism behind CXB and PIP mediated cell death was explored. The co-treatment led to reactive oxygen species generation, mitochondrial dysfunction, caspase activation and enhanced apoptosis in HT-29 cells. Additionally, the combination treatment synergistically modulated Wnt/ß-catenin pathway, downregulated the stemness markers and boosted therapeutic response in CT26 syngeneic Balb/c mice. CONCLUSION: The outcomes of the study suggests that combining CXB and PIP offers a novel approach for the treatment of colon cancer.

CASC5 is a potential cancer-testis gene in human urinary bladder transitional cell carcinoma.

Urinary Bladder cancer (UBC) is a diversified disease with an array of clinicopathological attributes. Several studies have shown that cancer susceptibility candidate 5 (CASC5) plays important roles in various types of malignancies; however its expression and clinical significance in human UBC remain largely unknown. This research study was intended to explore mRNA/protein expression pattern of CASC5 as a member of the cancer-testis (CT) gene family and assess its clinical utility in diagnostic management of patients with UBC. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) was employed to appraise the detailed expression profile of CASC5 in patients with UBC. The mRNA over expression of CASC5 was detected in testis tissue and relatively high frequency 59.2% (45 of 76) of CASC5 mRNA was detected in UBC tissues. CASC5 mRNA relative mean fold expression was also significantly (p < 0.01) higher in the muscle-invasive tumor tissues compared to non-muscle-invasive tumor tissues (12.26 ± 9.53 vs. 4.64 ± 2.50, p = 0.005). Heterogeneous staining pattern of CASC5 protein was exclusively detected using IHC. The frequency of CASC5 protein over expression was detected in 67.7% (44 of 65) UBC patients and negative in benign prostatic hyperplasia (BPH). Further, CASC5 protein expression was significantly (p < 0.001) associated with cigarette smoking habit in UBC patients. Our study findings testified that CASC5 over expression among patients with UBC as compared to controls and concludes that CASC5 is a potential CT gene in UBC.

N-acetyl-cysteine in combination with celecoxib inhibits Deoxynivalenol induced skin tumor initiation via induction of autophagic pathways in swiss mice.

Deoxynivalenol is a trichothecene mycotoxin which naturally contaminates small grain, cereals intended for human and animal consumption. Investigations for dermal toxicity of DON has been needed and highlighted by WHO. Previous studies on dermal toxicity suggest that DON has DNA damaging potential leading to skin tumor initiation in mice skin. However, considering its toxicological manifestations arising after dermal exposure, strategies for its prevention/protection are barely available in literatute. Collectively, our study demonstrated that N-acetylcysteine (NAC), precursor of glutathione, significantly alters the genotoxic potential of DON. Further NAC in combination with Celecoxib (CXB) inhibits tumor growth by altering antioxidant status and increasing autophagy in DON initiated Swiss mice. Despite the broad spectrum use of CXB, its use is limited by the concerns about its adverse effects on the cardiovascular system. Serum parameters and histology analysis revealed that CXB (2 mg) when applied topically for 24 weeks did not impart any cardiovascular toxicity which could be because skin permeation potential of CXB was quite low when analyzed through HPLC analysis. Although the anticancer effects of CXB and NAC have been studied, however, the combination of NAC and CXB has yet not been explored for any cancer treatment. Therefore our observations provide additional insights into the therapeutic effects of combinatorial treatment of CXB and NAC against skin tumor prevention. This approach might form a novel alternative strategy for skin cancer treatment as well as skin associated toxicities caused by mycotoxins such as DON. This combinatorial approach can overcome the limitations associated with the use of CXB for long term as topical application of the same seems to be safe in comparison to the oral mode of administration.

Multifunctional hybrid nanoconstructs facilitate intracellular localization of doxorubicin and genistein to enhance apoptotic and anti-angiogenic efficacy in breast adenocarcinoma.

The progressive development of tumors leading to angiogenesis marks the advancement of cancer which requires specific targeted treatment preferably with combination chemotherapy. However, there is still a long way to go to develop an efficient delivery system that could overcome the tumor microenvironment to achieve efficient delivery. Therefore, we have developed spermine (SPM) tethered lipo-polymeric hybrid nanoconstructs with cell surface heparan sulfate proteoglycan (HSPG) specificity for higher intracellular localization and pH dependent charge reversal in the tumor microenvironment (below pH 5.8) to facilitate Doxorubicin (Dox) and Genistein (Gen) release in a synergistic combination. We have observed the specific uptake of SPM anchored hybrid nanoconstructs by receptor-mediated endocytosis in human breast cancer cells (MDA-MB-231) through the HSPG receptor. The SPM-D + G/NPs induced a higher rate of apoptosis in MDA-MB-231 cells via disruption of the mitochondrial membrane potential and also exhibited a stronger anti-angiogenic effect governing the inhibition of VEGF pathway modulation, proliferation, invasion and migration of HUVECs in in vitro and in vivo Balb/c mouse models. The involvement of Akt/Hif1α/VEGF dependent signal cascading and its down-regulation with a pro-apoptotic drug Dox and an anti-angiogenic agent Gen was evident as demonstrated by an in silico docking study and subsequently proven by RT-PCR and western blotting. Altogether this study highlights the potential role of SPM in targeting HSPG receptors and synergistic delivery of Dox and Gen as a promising strategy to effectively inhibit BAC progression and these findings could open a new window to deliver combinations of chemotherapeutic agents along with anti-angiogenic ligands using hybrid nanoparticles.

Induction of Mitochondrial Cell Death and Reversal of Anticancer Drug Resistance via Nanocarriers Composed of a Triphenylphosphonium Derivative of Tocopheryl Polyethylene Glycol Succinate.

We have devised a nanocarrier using "tocopheryl polyethylene glycol succinate (TPGS) conjugated to triphenylphosphonium cation" (TPP-TPGS) for improving the efficacy of doxorubicin hydrochloride (DOX). Triphenylphosphonium cation (TPP) has affinity for an elevated transmembrane potential gradient (mitochondrial), which is usually high in cancer cells. Consequently, when tested in molecular docking and cytotoxicity assays, TPP-TPGS, owing to its structural similarity to mitochondrially directed anticancer compounds of the "tocopheryl succinate" family, interferes specifically in mitochondrial CII enzyme activity, increases intracellular oxidative stress, and induces apoptosis in breast cancer cells. DOX loaded nanocarrier (DTPP-TPGS) constructed using TPP-TPGS was positively charged, spherical in shape, sized below 100 nm, and had its drug content distributed evenly. DTPP-TPGS offers greater intracellular drug delivery due to its rapid endocytosis and subsequent endosomal escape. DTPP-TPGS also efficiently inhibits efflux transporter P glycoprotein (PgP), which, along with greater cell uptake and inherent cytotoxic activity of the construction material (TPP-TPGS), cumulatively results in 3-fold increment in anticancer activity of DOX in resistant breast cancer cells as well as greater induction of necroapoptosis and arrest in all phases of the cell cycle. DTPP-TPGS after intravenous administration in Balb/C mice with breast cancer accumulates preferentially in tumor tissue, which produces significantly greater antitumor activity when compared to DOX solution. Toxicity evaluation was also performed to confirm the safety of this formulation. Overall TPP-TPGS is a promising candidate for delivery of DOX.

Salinomycin inhibits breast cancer progression via targeting HIF-1α/VEGF mediated tumor angiogenesis in vitro and in vivo.

Cancer is a complex disease wherein cells begin to divideabnormally and spread into surrounding tissues. Angiogenesis plays a crucial role in tumor progression as it is required for sustained growth and metastasis, therefore targeting angiogenesis is a promising therapeutic approach for breast cancer management. Salinomycin (SAL) has been reported to exhibit anticancer response on various types of cancer. In the present study, we explored the antiangiogenic and anticancer efficacy of the polyether ionophore SAL in the breast cancer model. It effectively inhibited cell proliferation, invasion, and migration. It also inhibited the expression of pro-angiogenic cell surface marker CD31 in HUVEC, thereby interrupting the endothelial tubulogenesis. It decreased the HIF-1α transcription factor DNA binding activity to HRE sequence in HUVEC and human breast cancer cells. Further, corresponding to our in vitro findings, SAL suppressed neovascularization in the chick chorioallantoic membrane and the Matrigel plug implanted mice model. Bioluminescence and immunofluorescence imaging revealed that SAL treatment in mice inhibits breast cancer growth and tumor angiogenesis. SAL also suppressed the serum VEGFA level in tumor-bearing mice and induced caspase-dependent apoptosis in breast cancer cells. Taken together our findings suggested that SAL inhibits VEGF induced angiogenesis and breast cancer growth via interrupting HIF-1α/VEGF signalling and could be used as a promising antiangiogenic agent for breast cancer treatment.

Safety assessment of the pharmacological excipient, diethylene glycol monoethyl ether (DEGEE), using in vitro and in vivo systems.

BACKGROUND: Diethylene glycol monoethyl ether (DEGEE) is widely used as a solubilizer in cosmetics as well as in oral, topical, transdermal and injectable pharmaceutical formulations. Due to the unavailability of detailed toxicological studies on DEGEE, the Scientific Committee on Consumer Products (SCCP) found its toxicological reports to be unsatisfactory, comprising only summaries. Also, a few reports have raised concern on the use of DEGEE as it might cause damage to the kidneys. OBJECTIVE: Safety assessment of DEGEE using in vitro and in vivo models. METHODS: In vitro effects of DEGEE (0.5-25 mg/ml) were assessed in the HEK293 human embryonic kidney cells. In vivo effects were evaluated after single acute exposure of DEGEE via intraperitoneal route in Swiss albino mice and further, a 28 days subchronic exposure study was conducted where DEGEE was administered orally, once daily. RESULTS: DEGEE was cytotoxic to HEK293 cells, and an IC50 of 15 mg/ml was established. An increase in the intracellular levels of ROS and alteration in the mitochondrial membrane potential led to nuclear fragmentation and induction of apoptosis in these cells. Survival rate of animals administered intraperitoneally with a single acute dose of 1000 mg/kg DEGEE was 100% with no significant changes in the behavioural and histological parameters. However, the dose of 3000 mg/kg and above led to total mortality within 14 days of acute exposure. Subchronic oral exposure of 500-2000 mg/kg DEGEE showed no significant changes in the hematological, biochemical and histopathological parameters. CONCLUSIONS: The in vitro findings indicate that the nephrotoxic potential of DEGEE cannot be ruled out. The results of the in vivo studies reveal that the degree of toxic effects shown by DEGEE varies, depending on the dose, duration of exposure and routes of administration. Therefore, the present findings are of relevance and thorough studies should be conducted before using this substance in clinical formulations. Graphical abstract Evaluation of the toxic potential of Diethylene glycol monoethyl ether.

A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasiveness†.

Endometriosis is a prevalent gynecological disorder that eventually gives rise to painful invasive lesions. Increased levels of transforming growth factor-beta 1 (TGF-B1) have been reported in endometriosis. However, details of the effects of high TGF-B1 on downstream signaling in ectopic endometrial tissue remain obscure. We induced endometriotic lesions in mice by surgical auto-transplantation of endometrial tissues to the peritoneal regions. We then treated endometriotic (ectopic and eutopic endometrial tissues) and nonendometriotic (only eutopic endometrial tissues) animal groups with either active TGF-B1 or PBS. Our results demonstrate that externally supplemented TGF-B1 increases the growth of ectopically implanted endometrial tissues in mice, possibly via SMAD2/3 activation and PTEN suppression. Adhesion molecules integrins (beta3 and beta8) and FAK were upregulated in the ectopic endometrial tissue when TGF-B1 was administered. Phosphorylated E-cadherin, N-cadherin, and vimentin were enhanced in the ectopic endometrial tissue in the presence of TGF-B1 in the mouse model, and correlated with epithelial-mesenchymal transition (EMT) in ovarian endometriotic cells of human origin. Furthermore, in response to TGF-B1, the expression of RHOGTPases (RAC1, RHOC, and RHOG) was increased in the human endometriotic cells (ovarian cyst derived cells from endometriosis patient) and tissues from the mouse model of endometriosis (ectopic endometrial tissue). TGF-B1 enhanced the migratory, invasive, and colonizing potential of human endometriotic cells. Therefore, we conclude that TGF-B1 potentiates the adhesion of ectopic endometrial cells/tissues in the peritoneal region by enhancing the integrin and FAK signaling axis, and also migration via cadherin-mediated EMT and RHOGTPase signaling cascades.

Synchronized Ratiometric Codelivery of Metformin and Topotecan through Engineered Nanocarrier Facilitates In Vivo Synergistic Precision Levels at Tumor Site.

The combination of metabolic modulators with chemotherapy holds vast promise for effective inhibition of tumor progression and invasion. Herein, a ratiometric codelivery platform is developed for metformin (MET), a known metabolic modulator and topotecan (TPT), a chemotherapeutic drug, by engineering lipid bilayer-camouflaged mesoporous silica nanoparticles (LB-MSNs). In an attempt to deliver and maintain high tumor site concentrations of MET and TPT, a novel ion pairing-assisted loading procedure is developed using pamoic acid (PA) as an in situ trapping agent. PA, a hydrophobic counterion, increases the hydrophobicity of MET and TPT and facilitates MSNs with exceptionally high payload capacity (>40 and 32 wt%, respectively) and controlled release profile. Further, the synergy between MET and TPT determined by a modeling approach helps to afford synchronized delivery of both the drugs. Coloaded MET and TPT LB-MSNs present synergistic cytotoxicity against MDA-MB-231/4T1 cells and effectively promote apoptosis via mitochondrial membrane depolarization and cell cycle arrest. Extended pharmacokinetic profiles in preclinical models with fourfold to sevenfold longer circulation half-life and 7.5-100 times higher tumor site concentrations correspond to a significant increase in pharmacodynamic efficacy. Taken together, the developed codelivery approach effectively addresses the challenges in the chemotherapeutic efficacy of MET and TPT collectively.

Anisamide-Anchored Lyotropic Nano-Liquid Crystalline Particles with AIE Effect: A Smart Optical Beacon for Tumor Imaging and Therapy.

The prospective design of nanocarriers for personalized oncotherapy should be an ensemble of targeting, imaging, and noninvasive therapeutic capabilities. Herein, we report the development of the inverse hexagonal nano-liquid crystalline (NLC) particles that are able to host formononetin (FMN), a phytoestrogen with known anticancer activity, and tetraphenylethene (TPE), an iconic optical beacon with aggregation-induced emission (AIE) signature, simultaneously. Ordered three-dimensional mesoporous internal structure and high-lipid-volume fraction of NLC nanoparticles (NLC NPs) frame the outer compartment for the better settlement of payloads. Embellishment of these nanoparticles by anisamide (AA), a novel sigma receptor targeting ligand using carbodiimide coupling chemistry ensured NLC's as an outstanding vehicle for possible utility in surveillance of tumor location as well as the FMN delivery through active AIE imaging. The size and structural integrity of nanoparticles were evaluated by quasi-elastic light scattering, cryo field emission scanning electron microscopy small-angle X-ray scattering. The existence of AIE effect in the nanoparticles was evidenced through the photophysical studies that advocate the application of NLC NPs in fluorescence-based bioimaging. Moreover, confocal microscopy illustrated the single living cell imaging ability endowed by the NLC NPs. In vitro and in vivo studies supported the enhanced efficacy of targeted nanoparticles (AA-NLC-TF) in comparison to nontargeted nanoparticles (NLC-TF) and free drug. Apparently, this critically designed multimodal NLC NPs may establish a promising platform for targeted and image-guided chemotherapy for breast cancer.

Isoniazid induces apoptosis: Role of oxidative stress and inhibition of nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2).

AIMS: Long-term treatment of Isoniazid (INH) in tuberculosis (TB) patients can lead to anti-tuberculosis drug-induced hepatotoxicity. To understand the mechanism of hepatotoxicity, an attempt has been made to elucidate the role of Nrf2, a transcription factor induced by oxidative stress, in INH induced apoptosis liver cancer cell lines. MATERIALS AND METHODS: Cytotoxicity was evaluated by MTT assay. Apoptosis and reactive oxygen species (ROS) generation was performed by flow cytometry. mRNA and protein expression of various genes involves in INH induced toxicity was evaluated via Real-time PCR and western blot analysis respectively. Differential protein expression was performed by two-dimensional gel electrophoresis followed by identification using MALDI TOF/TOF. KEY FINDINGS: INH induced ROS and apoptosis in HepG2 as well as THLE-2 cells. Nuclear damage was also observed by INH treatment in HepG2 cells. Expression of apoptotic (Cytochrome C and Caspase 9) and antioxidative (Keap1 and Nrf2) genes were observed to increase. INH induced PKCδ phosphorylation and released Nrf2 from its inhibitor Keap1 in the cytoplasm of HepG2 cells. However, over-expression of Nrf2 did not affect nuclear Nrf2 protein level as well as its downstream target NQO1. Nrf2 importer, Karyopherin ß1 level was observed to decrease in HepG2 as well as THLE-2 cells following INH treatment. SIGNIFICANCE: These findings suggest that INH prevented Nrf2 translocation into the nucleus by inhibiting its importer Karyopherin ß1. Therefore Nrf2 might not able to bind ARE sequences from inducing antioxidative response for protecting the cells undergoing apoptosis.

Centchroman regulates breast cancer angiogenesis via inhibition of HIF-1α/VEGFR2 signalling axis.

AIMS: Angiogenesis is a recognized hallmark of cancer which promotes cancer cell progression and metastasis. Inhibition of angiogenesis to attenuate cancer growth is becoming desirable strategy for breast cancer management. The present study is aimed to investigate the antiangiogenic efficacy of a novel selective estrogen receptor modulator Centchroman (CC) on human breast cancer cells. MAIN METHODS: Effect of CC on cell viability was evaluated using Sulforhodamine B assay. Endothelial cell proliferation, wound healing, Boyden chamber cell invasion, tube formation and chorioallantoic membrane (CAM) assays were performed to assess the effect of CC on migration, invasion and angiogenesis. Apoptosis, reactive oxygen species generation, caspase-3/7 and intracellular calcium ion level were measured through flow cytometry. Expression levels of HIF-1α, VEGF, VEGFR2, AKT and ERK were assessed by western blot analysis. KEY FINDINGS: CC selectively induces apoptosis in human breast cancer cells without affecting non-tumorigenic breast epithelial cells MCF-10A. Moreover, it inhibits migratory, invasive and mammosphere forming potential of breast cancer. Furthermore, CC also inhibited VEGF-induced migration, invasion and tube formation of HUVECs in vitro. CC effectively inhibited neovasculature formation in chicken CAM. Western blot analysis demonstrated that CC inhibited expression of HIF-1α and its downstream target VEGF. Interestingly, CC also suppressed VEGFR2 phosphorylation and consequently attenuated AKT and ERK phosphorylation. SIGNIFICANCE: Our findings suggest that CC downregulates VEGF-induced angiogenesis by modulating HIF-1α/VEGFR2 pathway and recommend it (CC) as a potential therapeutic drug for breast cancer treatment.

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction.

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca2+ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.

miRNA genetic variants: As potential diagnostic biomarkers for oral cancer.

MicroRNAs (miRNAs) comprise a novel class of small, non-coding endogenous RNAs that have a role in the plethora of regulatory activities by directing their target mRNAs for degradation or translational repression. Single nucleotide polymorphisms (SNPs) in miRNA genes can lead to alteration in mRNA expression, resulting in serious consequences. Detection of miRNA-polymorphisms can potentially improve diagnosis, treatment, prognosis in patients and has extreme implications in the fields of pharmacogenomics and personalization of medicine. The aim of this study is to investigate the association between miR-499 A/G and miR-149 C/T polymorphisms with susceptibility to development of Oral Squamous Cell Carcinoma (OSCC). 200 histopathologically diagnosed and confirmed samples from OSCC patients and 200 control samples from the general population were recruited for the study. All OSCC patients were graded based on their differentiation, and genetic analysis was performed by PCR-RFLP and sequencing. qRT-PCR was carried out to compare the expression of miR-499 and miR-149 in different grades of various stages of OSCC patients concerning to the controls. Further Immunohistochemistry (IHC) was performed to study the target gene of miR-499. The study shows a probable association of miR-499 A/G and miR-149 C/T with susceptibility of OSCC. Random sequencing analysis and Immunohistochemistry contribute to the result that miR-499 A/G increases the susceptibility of OSCC by targeting SOX-6. PCR- Restriction Fragment Length Polymorphism (RFLP) and multivariable logistic analysis revealed that there is a significant association between miR-149 CT+TT and CT and susceptibility of OSCC. Our study suggests that miR-499 A/G and miR-149 C/T polymorphisms may play crucial roles in susceptibility and development of OSCC in Indian population.

Novel combination of salinomycin and resveratrol synergistically enhances the anti-proliferative and pro-apoptotic effects on human breast cancer cells.

Resveratrol (RES) is a natural polyphenol having anti-proliferative activity against breast cancer cells. RES in combination with other chemo modulatory agents, minimizes toxicity and increases efficacy of the treatment. Salinomycin (SAL), a monocarboxylic polyether ionophore is known for selectively targeting breast cancer stem cells. Purpose of the present study was to investigate whether RES in combination with SAL exerts synergistic anti-proliferative activity on breast cancer cells. We further evaluated the molecular mechanism behind SAL and RES mediated cell death. Cytotoxicity assay was performed to determine 50% inhibitory concentration (IC50) of SAL and RES in different human breast cancer cells (HBCCs). Drug synergism and combination index (CI) were calculated using CompuSyn software and effects of synergistic combinations (CI < 1) involving lower doses of SAL and RES were selected for further studies. This combination significantly induced apoptosis in HBCCs without affecting non tumorigenic human breast epithelial cells MCF-10A. Co-treatment enhanced apoptosis in MCF-7 cells via reactive oxygen species (ROS) mediated mitochondrial dysfunction. Oxidative stress disrupt redox homeostasis which altered antioxidant enzymes viz. CuZn Superoxide dismutase (SOD), MnSOD and catalase. Additionally, combination altered nuclear morphology, enhanced PARP cleavage and led to caspase activation. SAL and RES also synergistically modulated MAPK pathway. Study suggests that SAL and RES offer a novel combination approach for the treatment of breast cancer.