The role of endolysosomal trafficking in anticancer drug resistance.

Multidrug resistance (MDR) remains a major obstacle towards curative treatment of cancer. Despite considerable progress in delineating the basis of intrinsic and acquired MDR, the underlying molecular mechanisms remain to be elucidated. Emerging evidences suggest that dysregulation in endolysosomal compartments is involved in mediating MDR through multiple mechanisms, such as alterations in endosomes, lysosomes and autophagosomes, that traffic and biodegrade the molecular cargo through macropinocytosis, autophagy and endocytosis. For example, altered lysosomal pH, in combination with transcription factor EB (TFEB)-mediated lysosomal biogenesis, increases the sequestration of hydrophobic anti-cancer drugs that are weak bases, thereby producing an insufficient and off-target accumulation of anti-cancer drugs in MDR cancer cells. Thus, the use of well-tolerated, alkalinizing compounds that selectively block Vacuolar H⁺-ATPase (V-ATPase) may be an important strategy to overcome MDR in cancer cells and increase chemotherapeutic efficacy. Other mechanisms of endolysosomal-mediated drug resistance include increases in the expression of lysosomal proteases and cathepsins that are involved in mediating carcinogenesis and chemoresistance. Therefore, blocking the trafficking and maturation of lysosomal proteases or direct inhibition of cathepsin activity in the cytosol may represent novel therapeutic modalities to overcome MDR. Furthermore, endolysosomal compartments involved in catabolic pathways, such as macropinocytosis and autophagy, are also shown to be involved in the development of MDR. Here, we review the role of endolysosomal trafficking in MDR development and discuss how targeting endolysosomal pathways could emerge as a new therapeutic strategy to overcome chemoresistance in cancer.

A Novel Dialkylamino-Functionalized Chalcone, DML6, Inhibits Cervical Cancer Cell Proliferation, In Vitro, via Induction of Oxidative Stress, Intrinsic Apoptosis and Mitotic Catastrophe.

In this study, we designed, synthesized and evaluated, in vitro, novel chalcone analogs containing dialkylamino pharmacophores in the cervical cancer cell line, OV2008. The compound, DML6 was selective and significantly decreased the proliferation of OV2008 and HeLa cells in sub-micromolar concentrations, compared to prostate, lung, colon, breast or human embryonic kidney cell line (HEK293). DML6, at 5 µM, arrested the OV2008 cells in the G2 phase. Furthermore, DML6, at 5 µM, increased the levels of reactive oxygen species and induced a collapse in the mitochondrial membrane potential, compared to OV2008 cells incubated with a vehicle. DML6, at 5 µM, induced intrinsic apoptosis by significantly (1) increasing the levels of the pro-apoptotic proteins, Bak and Bax, and (2) decreasing the levels of l the anti-apoptotic protein, Bcl-2, compared to cell incubated with a vehicle. Furthermore, DML6, at 5 and 20 µM, induced the cleavage of caspase-9, followed by subsequent cleavage of the executioner caspases, caspase-3 and caspase-7, which produced OV2008 cell death. Overall, our data suggest that DML6 is an apoptosis-inducing compound that should undergo further evaluation as a potential treatment for cervical cancer.

Antiproliferative Efficacy of N-(3-chloro-4-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine, DW-8, in Colon Cancer Cells Is Mediated by Intrinsic Apoptosis.

A novel series of 4-anilinoquinazoline analogues, DW (1-10), were evaluated for anticancer efficacy in human breast cancer (BT-20) and human colorectal cancer (CRC) cell lines (HCT116, HT29, and SW620). The compound, DW-8, had the highest anticancer efficacy and selectivity in the colorectal cancer cell lines, HCT116, HT29, and SW620, with IC50 values of 8.50 ± 2.53 µM, 5.80 ± 0.92 µM, and 6.15 ± 0.37 µM, respectively, compared to the non-cancerous colon cell line, CRL1459, with an IC50 of 14.05 ± 0.37 µM. The selectivity index of DW-8 was >2-fold in colon cancer cells incubated with vehicle. We further determined the mechanisms of cell death induced by DW-8 in SW620 CRC cancer cells. DW-8 (10 and 30 µM) induced apoptosis by (1) producing cell cycle arrest at the G2 phase; (2) activating the intrinsic apoptotic pathway, as indicated by the activation of caspase-9 and the executioner caspases-3 and 7; (3) nuclear fragmentation and (4) increasing the levels of reactive oxygen species (ROS). Overall, our results suggest that DW-8 may represent a suitable lead for developing novel compounds to treat CRC.

Novel Chrysin-De-Allyl PAC-1 Hybrid Analogues as Anticancer Compounds: Design, Synthesis, and Biological Evaluation.

New chrysin-De-allyl-Pac-1 hybrid analogues, tethered with variable heterocyclic systems (4a-4o), were rationally designed and synthesized. The target compounds were screened for in vitro antiproliferative efficacy in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and normal human mammary epithelial cells (HMECs). Two compounds, 4g and 4i, had the highest efficacy and selectivity towards MDA-MB-231 cells, and thus, were further evaluated by mechanistic experiments. The results indicated that both compounds 4g and 4i induced apoptosis by (1) inducing cell cycle arrest at the G2 phase in MDA-MB-231 cells, and (2) activating the intrinsic apoptotic pathways in a concentration-dependent manner. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. Overall, our results suggest that 4g and 4i could be suitable leads for developing novel compounds to treat TNBC.

Ethnic disparities in the immune microenvironment of triple negative breast cancer and its role in therapeutic outcomes.

In 2020, newly diagnosed breast cancer (BC) cases surpassed that of lung cancer among women, making it the most common female cancer globally. In spite of recent increases in incidence rates, mortality due to BC has declined since 1989. These declines have been attributed to advancements in treatment modalities as well as increased mammography surveillance. Despite these advances, African American (AA) women are 40% more likely to die from BC than Caucasian women. Multifactorial etiology has been implicated in the disparity of BC mortality rates among AA women. As an example, AA women have a disproportionate incidence of triple negative breast cancer (TNBC), which has a poor prognosis and marginal treatment options. Increasingly, the tumor microenvironment (TME) has gained relevance as it relates to primary tumor progression, metastasis and treatment possibilities. The treatment outcomes or pathological complete response (pCR) in TNBC among AA women are affected by differences in TME. The TME of AA women exhibit several variances in acellular and cellular components associated with pro-tumorigenic effects. For example, increased levels of the adipocyte-related hormone, resistin, the pro-inflammatory cytokine, IL-6, and the CC chemokine, CCL2, within the TME of AA women gives rise to an increased density of M2 macrophages, also known as tumor-associated macrophages. Elevated levels of vascular endothelial growth factor in the TME of AA women increase the vascular density or vascularity, which facilitate aggressive tumor growth and metastasis. Furthermore, a pro-tumorigenic TME is supported by increased levels of the CXC chemokine, CXCL12 that results in the recruitment of regulatory T lymphocytes (Tregs ). Due to these and other differences in the TME of AA women, precision oncology can target specific aspects of the TME that may contribute to a poorer prognosis. In addition to the discrepancies in the TME, AA women face socio-economic barriers that limit their ability to access state-of-the-art, novel therapies against metastatic TNBC. In this review, we will provide a brief overview of the tumor immune microenvironment, immune-based treatment options for TNBC and their potential to decrease health disparities due to ethnicity.

IND-2, a Quinoline Derivative, Inhibits the Proliferation of Prostate Cancer Cells by Inducing Oxidative Stress, Apoptosis and Inhibiting Topoisomerase II.

In men, prostate cancer (PC) is the most frequently diagnosed cancer, causing an estimated 375,000 deaths globally. Currently, existing therapies for the treatment of PC, notably metastatic cases, have limited efficacy due to drug resistance and problematic adverse effects. Therefore, it is imperative to discover and develop novel drugs for treating PC that are efficacious and do not produce intolerable adverse or toxic effects. Condensed quinolines are naturally occurring anticancer compounds. In this study, we determined the in vitro efficacy of IND-2 (4-chloro-2-methylpyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolone) in the PC lines, PC-3 and DU-145. IND-2 significantly inhibited the proliferation of PC-3 and DU-145, with IC50 values of 3 µM and 3.5 µM, respectively. The incubation of PC-3 cells with 5 and 10 µM of IND-2 caused the loss of the mitochondrial membrane potential in PC-3 cells. Furthermore, IND-2, at 5 µM, increased the expression of cleaved caspase-3, cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase (PARP). The incubation of PC-3 cells with 5 µM of IND-2 significantly decreased the expression of the apoptotic protein, B-cell lymphoma 2 (Bcl-2). Furthermore, 5 and 10 µM of IND-2 produced morphological changes in PC-3 cells characteristic of apoptosis. Interestingly, IND-2 (2.5, 5 and 10 µM) also induced mitotic catastrophe in PC-3 cells, characterized by the accumulation of multinuclei. The incubation of DU-145 cells with 1.25 and 5 µM of IND-2 significantly increased the levels of reactive oxygen species (ROS). Finally, IND-2, at 10 µM, inhibited the catalytic activity of topoisomerase IIα. Overall, our findings suggest that IND-2 could be a potential lead compound for the development of more efficacious compounds for the treatment of PC.

Automated detection of apoptotic versus nonapoptotic cell death using label-free computational microscopy.

Identification of cell death mechanisms, particularly distinguishing between apoptotic versus nonapoptotic pathways, is of paramount importance for a wide range of applications related to cell signaling, interaction with pathogens, therapeutic processes, drug discovery, drug resistance, and even pathogenesis of diseases like cancers and neurogenerative disease among others. Here, we present a novel high-throughput method of identifying apoptotic versus necrotic versus other nonapoptotic cell death processes, based on lensless digital holography. This method relies on identification of the temporal changes in the morphological features of mammalian cells, which are unique to each cell death processes. Different cell death processes were induced by known cytotoxic agents. A deep learning-based approach was used to automatically classify the cell death mechanism (apoptotic vs necrotic vs nonapoptotic) with more than 93% accuracy. This label free approach can provide a low cost (<$250) alternative to some of the currently available high content imaging-based screening tools.

A Novel Thienopyrimidine Analog, TPH104, Mediates Immunogenic Cell Death in Triple-Negative Breast Cancer Cells.

Enhancing the tumor immunogenic microenvironment has been suggested to circumvent triple-negative breast cancer (TNBC) resistance and increase the efficacy of conventional chemotherapy. Here, we report a novel chemotherapeutic compound, TPH104, which induces immunogenic cell death in the TNBC cell line MDA-MB-231, by increasing the stimulatory capacity of dendritic cells (DCs), with an IC50 value of 140 nM. TPH104 (5 µM) significantly increased ATP levels in the supernatant and mobilized intracellular calreticulin to the plasma membrane in MDA-MB-231 cells, compared to cells incubated with the vehicle. Incubating MDA-MB-231 cells for 12 h with TPH104 (1-5 µM) significantly increased TNF-α mRNA levels. The supernatants of dying MDAMB-231 cells incubated with TPH104 increased mouse bone marrow-derived DC maturation, the expression of MHC-II and CD86 and the mRNA expression of TNF-α, IL-6 and IL-12. Overall, these results indicate that TPH104 induces immunogenic cell death in TNBC cells, in part, by activating DCs.