Adapalene and Doxorubicin Synergistically Promote Apoptosis of TNBC Cells by Hyperactivation of the ERK1/2 Pathway Through ROS Induction.

Doxorubicin is a commonly used chemotherapeutic agent to treat several malignancies, including aggressive tumors like triple-negative breast cancer. It has a limited therapeutic index owing to its extreme toxicity and the emergence of drug resistance. As a result, there is a pressing need to find innovative drugs that enhance the effectiveness of doxorubicin while minimizing its toxicity. The rationale of the present study is that combining emerging treatment agents or repurposed pharmaceuticals with doxorubicin might increase susceptibility to therapeutics and the subsequent establishment of improved pharmacological combinations for treating triple-negative breast cancer. Additionally, combined treatment will facilitate dosage reduction, reducing the toxicity associated with doxorubicin. Recently, the third-generation retinoid adapalene was reported as an effective anticancer agent in several malignancies. This study aimed to determine the anticancer activity of adapalene in TNBC cells and its effectiveness in combination with doxorubicin, and the mechanistic pathways in inhibiting tumorigenicity. Adapalene inhibits tumor cell growth and proliferation and acts synergistically with doxorubicin in inhibiting growth, colony formation, and migration of TNBC cells. Also, the combination of adapalene and doxorubicin enhanced the accumulation of reactive oxygen species triggering hyperphosphorylation of Erk1/2 and caspase-dependent apoptosis. Our results demonstrate that adapalene is a promising antitumor agent that may be used as a single agent or combined with present therapeutic regimens for TNBC treatment.

Role of GW182 protein in the cell.

GW182 proteins interact directly with the argonaute proteins and constitute key components of miRNA repressor complexes (miRISC) in metazoans. As argonautes are insufficient for silencing they recruit the GW182 s that act as scaffold proteins inducing downstream translational repression, target mRNA deadenylation and exonucleolytic mRNA degradation. Besides their role as part of repressor complexes inside the cell, they function in wide variety of cellular processes as highlighted in this review. The present review summarises and discusses in detail our current knowledge of the GW182 s and their role inside the cell.

Molecular Alterations and Expression Dynamics of LATS1 and LATS2 Genes in Non-Small-Cell Lung Carcinoma.

Large tumor suppressor (LATS) is an important member of the Hippo pathway which can regulate organ size and cell proliferation. However, very little is known about the expression and clinical significance of LATS in lung cancer especially from this part of the world. We elucidated the frequency of LATS1 &LATS2 promoter hypermethylation (by methylation-specific PCR) and expression (by real-time PCR) in sixty nine (n = 69) Non-Small Cell Lung Cancer (NSCLC) patients and their corresponding normal lung tissue samples. We found promoter hypermethylation frequencies of LATS1 & LATS1to be 66.66% (46/69) and 71% (49/69) in NSCLC tissues. Decreased LATS1 & LATS2 mRNA expression was found in 55% and 66.66% of NSCLC patients. The LATS1 mRNA expression was significantly higher in normal lung tissues. Also, the mRNA levels of LATS1 and LATS2 NSCLC tissues with hypermethylation were significantly lower. Multivariable analysis confirmed that LATS1 under expression increased the hazard of death after adjusting for other clinicopathological factors. Importantly, the loss of LATS1 mRNA expression was associated with overall short survival. LATS1 is an independent prognostic factor and may play an important role in NSCLC progression and may serve as a novel therapeutic target of NSCLC.

TAZ is an independent prognostic factor in non-small cell lung carcinoma: Elucidation at protein level.

BACKGROUND: Hippo-LATS pathway is involved in regulating multiple phenotypes. TAZ (transcriptional co-activator with PDZ-binding motif) is a prominent proto-oncogene of this developmental pathway. Elevated TAZ expression has been observed in many cancers including Non-Small Cell lung cancer (NSCLC). OBJECTIVE: We elucidated the frequency of protein expression of TAZ gene in NSCLC patients of our population along with its relationship with clinicopathological parameters and determined its prognostic significance concerning survival in patients with resected tumor. METHODS: We looked into the protein expression of TAZ gene in sixty nine (n= 69) cases of NSCLC tissue and their corresponding normal lung tissue samples by western blotting. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of TAZ protein expressionon survival. RESULTS: Here, we found that TAZ protein expression was elevated in 49.27% (34 of 69) of NSCLC tissues as compared to only 13.04% (09 of 69) in normal lung tissues. TAZ protein expression was significantly associated with smoking, positive lymph node status and vascular invasion (P< 0.05). TAZ protein overexpression increased the hazards ratio by 2.6 (P= 0.005) on univariate analysis. Multivariable analysis confirmed that TAZ protein overexpression along with age and lymph node metastasis increased the hazard of death after adjusting for other clinicopathological factors (P< 0.05). Importantly, TAZ protein overexpression was associated with short overall survival and disease-free survival when compared with normal TAZ expression. CONCLUSION: These results indicate that TAZ is an independent prognostic factor and plays an important role in NSCLC progression and may serve as a novel therapeutic target of NSCLC.

The carboxy-terminal domain of connexin 43 (CT-Cx43) modulates the expression of p53 by altering miR-125b expression in low-grade human breast cancers.

PURPOSE: Connexin 43 (Cx43) is a widely expressed gap junction protein. It can also regulate various gap-junction independent processes, including cellular proliferation. The latter regulatory functions have been attributed to its carboxy-terminal domain, CT-Cx43. CT-Cx43 has been found to be expressed independent of full-length Cx43 in various cell types. Its nuclear localization has additionally raised the possibility that it may regulate the expression of particular genes, including miRNAs, known play a role in the regulation of cellular proliferation. Here, we set out to uncover the molecular mechanism(s) underlying CT-Cx43 mediated gene (de-)regulation in human breast cancer. METHODS: Western blotting and quantitative real time PCR were carried to assess the expression of CT-Cx43 and miR-125b in a panel of 60 primary human breast cancer tissues and its paired normal adjacent tissues. In addition, CT-Cx43 was exogenously expressed in the breast cancer-derived cell line MCF-7 and its effect on the expression of miR-125b and its downstream target p53 were evaluated, as well as its effect on cellular proliferation and death using MTT and LDH assays, respectively. RESULTS: We found that CT-Cx43, but not full-length Cx43, was down-regulated in low grade human breast cancers. In addition, we found that the tumor suppressor protein p53 exhibited a decreased expression in the CT-Cx43 down-regulated samples. Interestingly, we found that miR-125b, a negative regulator of p53, exhibited an inverse expression relationship with CT-Cx43 in the breast cancer samples tested. This inverse relationship was confirmed by exogenous expression of CT-Cx43 in MCF-7 cells. In addition, we found that CT-Cx43 up-regulation and subsequent miR-125b down-regulation resulted in a decreased proliferation of MCF-7 cells. CONCLUSIONS: Our data suggest a mechanism by which CT-Cx43 may regulate cell proliferation. Targeting of CT-Cx43 and/or miR-125b may be instrumental for therapeutic intervention in human breast cancer.

Mutations in MicroRNA Genes and Their Binding Sites are Infrequently Associated with Human Colorectal Cancer in the Kashmiri Population.

MicroRNAs are small non-coding RNAs, 19-24 nucleotides in length that bind to the 3'UTR of target mRNAs and thus regulate gene expression post transcriptionally. MiRNAs have been implicated in various biological and pathological processes. The binding of miRNAs to 3'UTR is crucial for regulating the mRNA level and hence protein expression. The complementarity between the miRNA and its target mRNA is critical for the outcome of the miRNA mediated translational regulation. Changes in the nucleotide sequence of either the miRNA or its target binding site can deregulate gene expression and hence lead to the development of various pathological conditions, including tumorigenesis. To determine whether sequence alterations in miRNA genes and their target sites in mRNAs are associated with the colorectal cancers, we screened two miRNA genes-Let-7c, mir-206 and selected miRNA binding regions on KRAS, TP53 and GJA1 3'UTR. This study was carried out on 60 human colorectal cancer tissue samples. Our sequencing results did not reveal any mutation/single-nucleotide polymorphism in either the miRNAs or the miRNA binding sites in any of the tumor samples. This data suggests that mutations/SNPs targeting miRNA genes or their binding sites in 3'-untranslated regions are infrequent events in the development of colorectal cancer in Kashmiri population.

Protein kinase C (PKC) mediated interaction between conexin43 (Cx43) and K(+)(ATP) channel subunit (Kir6.1) in cardiomyocyte mitochondria: Implications in cytoprotection against hypoxia induced cell apoptosis.

PURPOSE OF RESEARCH: We have recently shown that adenosine-triphosphate-sensitive potassium [K(+)(ATP)] channel protein subunit, Kir6.1 is a phosphospecific interaction partner of the gap-junction protein connexin43 (Cx43). Since, both Cx43 and K(+)(ATP) are known to be involved in cell survival during hypoxia, we addressed the question, whether the interaction between Cx43 and K(+)(ATP) has a role in protecting cell against hypoxia-induced cell death. PRINCIPLE RESULTS: We report here that the Kir6.1 protein interacts, in a phosphospecific manner with Cx43 in the mitochondria of cardiomyocytic cell line H9C2. The hypoxia for 12-h resulted in the appreciable increase in the phosphorylation at the serine 262 (S262) of the Cx43 with the concomitant increase in the Cx43 and Kir6.1 interaction. Moreover, the increased interaction was mediated by a signaling pathway involving PKC and more specifically by PKC epsilon. Functional implications of the association between the Cx43 and Kir6.1 were found to prevent mitochondria mediated hypoxia induced cell apoptosis. MAJOR CONCLUSIONS: Our results demonstrate that PKC epsilon regulates the interaction between Cx43 and Kir6.1 in the cardiomyocyte mitochondria and this interaction prevents hypoxia induced cell death. Our results provide an interesting lead in developing effective strategies to protect cardiomyocytes from hypoxia/ischemia induced cell death.

Growth inhibition by bupivacaine is associated with inactivation of ribosomal protein S6 kinase 1.

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation.

Impact of molecular alterations of BRAF in the pathogenesis of thyroid cancer.

BRAF alterations represent a novel indicator of the progression and aggressiveness of thyroid carcinogenesis. So, the main aim of the study was to elucidate the involvement of BRAF gene mutations and its expression in Kashmiri (North India) patients and investigate their association with clinico-pathological characteristics. Mutational analysis of BRAF gene was performed by polymerase chain reaction followed by DNA sequencing, whereas analysis of BRAF protein expression was done by western blotting. Overall mutations in BRAF was found to be 25% (15 of 60) and all of them were transversions (T>A) affecting codon 600 (valine to glutamine), restricted only to papillary thyroid cancer and well-differentiated grade. Patients with well-differentiated disease and in particular elevated thyroid-stimulating hormone levels were significantly associated with BRAF mutations (P < 0.05). Overall, 90% (54 of 60) of thyroid cancer cases showed increased expression of BRAF and non-smokers being significantly associated with BRAF over-expression. Totally, 86.7% (13 of 15) of BRAF mutation-positive patients were having over-expression of BRAF protein, whereas 91.2% (41 of 45) of patients with wild-type BRAF status were having over-expressed BRAF protein (P > 0.05). We conclude that both mutational events as well as over-expression of BRAF gene is highly implicated in pathogenesis of thyroid cancer and the BRAF protein over-expression is independent of the BRAF mutational status of thyroid cancer patients.

The XRCC1 Arg399Gln gene polymorphism and risk of colorectal cancer: a study in Kashmir.

BACKGROUND: The DNA repair gene XRCC1 Arg399Gln gene polymorphism has been found to be implicated in the development of various cancers, including colorectal cancer (CRC), in different populations. We aimed to determine any association of this polymorphism with the risk of CRC in Kashmir. MATERIALS AND METHODS: A total of 120 confirmed cases of CRC and 146 healthy cancer free controls from the Kashmiri population were included in this study. Genotyping was carried out by the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Genotype frequencies of XRCC1 Arg399Gln observed in controls were 34.2%, 42.5% and 23.3% for GG (Arg/Arg), GA (Arg/Gln), AA( Gln/Gln), respectively, and 28.3%, 66.7% and 5% in cases, with an odds ratio (OR)=5.7 and 95% confidence interval (CI) =2.3-14.1 (p=0.0001). No significant association of Arg399Gln SNP with any clinicopathological parameters of CRC was found. CONCLUSIONS: We found the protective role of 399Gln allele against risk to the development of CRC. The XRCC1 heterozygote status appears to be a strong risk factor for CRC development in the Kashmiri population.

Novelty of Axin 2 and lack of Axin 1 gene mutation in colorectal cancer: a study in Kashmiri population.

Colorectal cancer is (CRC) one of the leading causes of mortality and morbidity. Various genetic factors have been reported to be involved in the development of colorectal cancers including Axin gene. Axin, a major scaffold protein, plays an important role in various bio signaling pathways. We aim to study mutational pattern of Axin gene in colorectal cancer patients of Kashmiri population. The paired tumor and adjacent normal tissue specimens of 50 consecutive patients with CRC were used in our study. The DNA preparations were evaluated for the occurrence of Axin 1 and Axin 2 gene mutations by direct DNA sequencing. We analyzed exon 1a, 1b, 1c, 2, 4, 6, and 10 of Axin 1 and exon 7 of Axin 2. In this study, we found a novel mutation of G>T (GCT>TCT) transversion in exon 7 of Axin 2 gene at codon G695T (p.alanine > serine) at a frequency of 6% (3/50). In the same exon of Axin 2 gene a single nucleotide polymorphism (SNP) was detected in codon L688L (CCT>CTT) at a frequency of 36% (18/50). In exon 1c of Axin 1 a SNP was detected at codon D726D (GAT>GAC) at a frequency of 62.5% (31/50). Both the SNPs were synonymous hence do not lead to change of amino acid. Although Axin 1 and Axin 2 gene mutations have been found to be involved in the development of colorectal cancers, it seems to be a relatively rare event in Kashmiri population. However, an interesting finding of this study is the novelty of Axin 2 gene mutations which may be a predisposing factor in ethnic Kashmiri population to CRC.

Diversity of axin in signaling pathways and its relation to colorectal cancer.

Colorectal cancer is one of the commonest cancers in western world, while majority of cases of CRC are sporadic, a significant minority occurs as a result of inherited genetic mutations. Various studies have demonstrated that tumorigenesis of colon arises as a result of accumulation of multiple genetic alterations targeting the single signal transduction pathway. Genetic alteration of axin has been implicated in different cancers including colorectal cancers. Axin being a multidomain protein interacts with multiple proteins and acts as major scaffold protein in signaling pathways like c-jun/SAPK, TGF-ß, and Wnt. In different signaling pathways, axin utilizes different domains and hence exerts distinct roles.