Identification of chemoresistance targets in doxorubicin-resistant lung adenocarcinoma cells using LC-MS/MS-based proteomics.

Acquired chemoresistance remains a significant challenge in the clinics as most of the treated cancers eventually emerge as hard-to-treat phenotypes. Therefore, identifying chemoresistance targets is highly warranted to manage the disease better. In this study, we employed a label-free LC-MS/MS-based quantitative proteomics analysis to identify potential targets and signaling pathways underlying acquired chemoresistance in a sub-cell line (A549DR) derived from the parental lung adenocarcinoma cells (A549) treated with gradually increasing doses of doxorubicin (DOX). Our proteomics analysis identified 146 upregulated and 129 downregulated targets in A549DR cells. The KEGG pathway and Gene ontology (GO) analysis of differentially expressed upregulated and downregulated proteins showed that most abundant upregulated pathways were related to metabolic pathways, cellular senescence, cell cycle, and p53 signaling. Meanwhile, the downregulated pathways were related to spliceosome, nucleotide metabolism, DNA replication, nucleotide excision repair, and nuclear-cytoplasmic transport. Further, STRING analysis of upregulated biological processes showed a protein-protein interaction (PPI) between CDK1, AKT2, SRC, STAT1, HDAC1, FDXR, FDX1, NPC1, ALDH2, GPx1, CDK4, and B2M, proteins. The identified proteins in this study might be the potential therapeutic targets for mitigating DOX resistance.

4-Methylbenzylidene camphor induced neurobehavioral toxicity in zebrafish (Danio rerio) embryos.

4-Methylbenzylidene camphor (4-MBC) is a widely used organic UV filter in personal care products. Extensive use of 4-MBC and its frequent detection in aquatic ecosystems defile the biota with muscular and neuronal impairments. This study investigates the neurobehavioral toxicity of 4-MBC using Danio rerio as a model organism. Embryos were exposed semi-statically to 4-MBC at 5, 50, and 500 µg/L concentrations for 10-day post fertilization (dpf). Embryos exhibited a significant thigmotaxis and decreased startle touch response with altered expression of nervous system mRNA transcripts on 5 & 10 dpf. Compared to the sham-exposed group, 4-MBC treated larvae exhibited changes in the expression of shha, ngn1, mbp, elavl3, α1-tubulin, syn2a, and gap43 genes. Since ngn1 induction is mediated by shh signaling during sensory neuron specification, the elevated protein expression of NGN1 indicates 4-MBC interference in the sonic hedgehog signaling pathway. This leads to sensory neuron impairment and function such as 'sense' as evident from reduced touch response. In addition, larval brain histology with a reduced number of cells in the Purkinje layer emblazing the defunct motor coordination. Predictive toxicity study also showed a higher affinity of 4-MBC to modeled Shh protein. Thus, the findings of the present work highlighted that 4-MBC is potential to induce developmental neurotoxicity at both behavioral and molecular functional perspectives, and developing D. rerio larvae could be considered as a suitable alternate animal model to assess the neurological dysfunction of organic UV filters.

Rifampicin-induced ER stress and excessive cytoplasmic vacuolization instigate hepatotoxicity via alternate programmed cell death paraptosis in vitro and in vivo.

AIMS: Rifampicin-induced hepatotoxicity is a primary cause of drug-induced liver injury (DILI), posing a significant challenge to its continued clinical application. Moreover, the mechanism underlying rifampicin-induced hepatotoxicity remains unclear. MAIN METHODS: Human hepatocyte line-17 (HHL-17) cells were treated with an increasing dose of rifampicin for 24 h, and male Wistar rats were given rifampicin [150 mg/kg body weight (bw)] orally for 28 days. Viability assay, protein expression, and cell death assays were analyzed in vitro. Moreover, liver serum markers, body/organ weight, H&E staining, protein expression, etc., were assayed in vivo. KEY FINDINGS: Rifampicin induced a dose-dependent hepatotoxicity in HHL-17 cells (IC50; 600 µM), and increased the serum levels of liver injury markers, e.g., alanine transaminase (ALT) and aspartate transaminase (AST) in rats. Rifampicin-induced cell death was non-apoptotic and non-necroptotic both in vitro and in vivo. Further, excessive cellular vacuolization and reduced expression of Alix protein confirmed the induction of paraptosis both in vitro and in vivo. In addition, a significant increase in the endoplasmic reticulum (ER) stress markers (e.g., BiP, CHOP, and total polyubiquitinated proteins) was detected, demonstrating the induction of ER stress and altered protein homeostasis. Interestingly, rifampicin-induced hepatotoxicity was associated with the inhibition of autophagy and enhanced reactive oxygen species (ROS) generation in HHL-17 cells. Furthermore, inhibition of protein synthesis by cycloheximide (CHX) suppressed paraptosis by alleviating rifampicin-induced ER stress and ROS generation. SIGNIFICANCE: Rifampicin-induced hepatotoxicity involves ER stress-driven paraptosis as a novel mechanism of its toxicity that may be targeted to protect liver cells from rifampicin toxicity.

Chloroquine synergizes doxorubicin efficacy in cervical cancer cells through flux impairment and down regulation of proteins involved in the fusion of autophagosomes to lysosomes.

Drug repurposing holds abundant opportunity in the development of novel anticancer drugs. Chloroquine (CQ), a FDA approved anti-malarial drug, is demonstrated to enhance anticancer efficacy of standard anticancer drugs including doxorubicin (DOX) in several types of cancer cells. Here, we aimed to exploit the chemosensitizing effects of CQ against DOX in human cervical cancer (HeLa) cells that remains to be investigated yet. We show that a combination of DOX (40 nM) and CQ (40 µM) resulted in a synergistic cytotoxicity (combination index; CI < 1) in HeLa cells compared to the DOX or CQ alone. Synergistic effect of the combination (DOX + CQ) was associated with the impaired autophagic flux and enhanced apoptosis. Following treatment with the combination (DOX + CQ), the level of p62/SQSTM and LC-3II proteins was increased, while a decrease was noted in the expression of LAMP-2, Syntaxin17, Rab 5, and Rab 7 proteins that play critical roles in the fusion of autophagosomes to lysosomes. Autophagy inhibition by combination (DOX + CQ) enhanced the apoptotic cell death synergistically by increasing the cleavage of procaspase-3 and PARP1. Further, a prior incubation of HeLa cells with Z-VAD-FMK (a pan-caspase inhibitor) for 4 h, suppressed the combination (DOX + CQ)-induced cell death. Our data suggest that a combination of DOX + CQ had a better anti-cancer efficacy in HeLa cells than either of the drugs alone. Thus, CQ, as a repurposed drug, may hold the potential to synergize anticancer effects of DOX in cervical cancer cells.

Bisphenol A exposure induces metastatic aggression in low metastatic MCF-7 cells via PGC-1α mediated mitochondrial biogenesis and epithelial-mesenchymal plasticity.

AIMS: The frequency of estrogen receptor alpha (ERα)-positive breast cancers and their metastatic progression is prevalent in females globally. Aberrant interaction of estrogen-like endocrine-disrupting chemicals (EDCs) is highly implicated in breast carcinogenesis. Studies have shown that single or acute exposures of weak EDCs such as bisphenol A (BPA) may not have a substantial pro-carcinogenic/metastatic effect. However, repeated exposure to EDCs is expected to strongly induce carcinogenic/metastatic progression, which remains to be studied. MAIN METHODS: Low metastatic ERα-positive human breast cancer cells (MCF-7) were exposed to nanomolar doses of BPA every 24 h (up to 200 days) to study the effect of repeated exposure on metastatic potential. Following the designated treatment of BPA, markers of epithelial-mesenchymal transition (EMT), migration and invasion, mitochondrial biogenesis, ATP levels, and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) knockdown assays were performed. KEY FINDINGS: A repeated exposure of low dose BPA induced stable epithelial-mesenchymal plasticity in MCF-7 cells to augment migration and invasion in the ERα-dependent pathway. Repeated exposures of BPA increased the levels of several mesenchymal markers such as N-cadherin, vimentin, cluster of differentiation 44 (CD44), slug, and alpha-smooth muscle actin (α-SMA), whereas reduced the level of E-cadherin drastically. BPA-induced mitochondrial biogenesis favored metastatic aggression by fulfilling bioenergetics demand via PGC-1α/NRF1/ERRα signaling. Knockdown of PGC-1α resulted in suppressing both mitochondrial biogenesis and EMT in BPA exposed MCF-7 cells. SIGNIFICANCE: Repeated exposures of low dose BPA may induce metastatic aggression in ERα-positive breast cancer cells via PGC-1α-mediated mitochondrial biogenesis and epithelial-mesenchymal plasticity.

Phytochemicals based chemopreventive and chemotherapeutic strategies and modern technologies to overcome limitations for better clinical applications.

Naturally occurring phytochemicals or plant derivatives are now being explored extensively for their health's benefits and medicinal uses. The therapeutic effect of phytochemicals has been reported in several pathophysiological settings such as inflammatory disorders, metabolic disorders, liver dysfunction, neurodegenerative disorders, and nephropathies. However, the most warranted therapeutic effects of phytochemicals were mapped to their anticancerous and chemopreventive action. Moreover, combining phytochemicals with standard chemotherapy has shown promising results in cancer therapy with minimal side effects and better efficacy. Many phytochemicals, like curcumin, resveratrol, and epigallocatechin-3-gallate, have been extensively investigated for their chemopreventive as well as chemotherapeutic effects. However, poor bioavailability, low solubility, hydrophobicity, and obscure target specificity restrict their therapeutic applications in the clinic. There has been a continually increasing interest to formulate nanoformulations of phytochemicals by using various nanocarriers, such as liposomes, micelles, nanoemulsions, and nanoparticles, to improve their bioavailability and target specificity, thereby maximizing the therapeutic potential. In the present review, we have summarized chemopreventive as well as chemotherapeutic action of some common phytochemicals and their major limitations in clinical application. Also, we have given an overview of strategies that can improve the efficacy of phytochemicals for their chemotherapeutic value in clinical settings.