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 reappraisal of the 6-O-desmethylnaproxen-sulfating activity of the human cytosolic sulfotransferases.

In this study, we aimed to obtain a comprehensive account of the human cytosolic sulfotransferases (SULTs) that are capable of sulfating 6-O-desmethylnaproxen (O-DMN), a major metabolite of naproxen. Of the 13 known human SULTs tested, 7 (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, and SULT1E1) displayed O-DMN-sulfating activity, when analyzed using an elevated substrate concentration (500 µmol·L-1) together with 14 µmol·L-1 of the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). At 10 µmol·L-1 O-DMN concentration, however, only SULT1A1 and SULT1A3 displayed detectable activity, with the former being nearly 2 orders of magnitude more active than the latter. A pH-dependence study indicated that SULT1A1 exhibited a broad pH optimum spanning pH 5.5-7. Kinetic parameters of the sulfation of O-DMN by SULT1A1 were determined. The production and release of sulfated O-DMN was demonstrated using cultured human HepG2 hepatoma cells and Caco-2 colon carcinoma cells. Moreover, assays using human organ specimens revealed that the O-DMN-sulfating activities present in the cytosols of liver and small intestine (at 502.5 and 497.2 pmol·min-1·(mg protein)-1, respectively) were much higher than those detected for the cytosols of lung and kidney. Taken together, these results provided relevant information concerning the sulfation of O-DMN both in vitro and in vivo.

Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis.

The inhibition of apoptosis, disruption of cellular microtubule dynamics, and over-activation of the epithelial mesenchymal transition (EMT), are involved in the progression, metastasis, and resistance of colorectal cancer (CRC) to chemotherapy. Therefore, the design of a molecule that can target these pathways could be an effective strategy to reverse CRC progression and metastasis. In this study, twelve novel silybin derivatives, HM015a-HM015k (15a-15k) and compound 17, were screened for cytotoxicity in CRC cell lines. Compounds HM015j and HM015k (15k and 15j) significantly decreased cell proliferation, inhibited colony formation, and produced cell cycle arrest in CRC cells. Furthermore, 15k significantly induced the formation of reactive oxygen species and apoptosis. It induced the cleavage of the intrinsic apoptotic protein (Bax p21) to its more efficacious fragment, p18. Compound 15k also inhibited tubulin expression and disrupted its structure. Compound 15k significantly decreased metastatic LOVO cell migration and invasion. Furthermore, 15k reversed mesenchymal morphology in HCT116 and LOVO cells. Additionally, 15k significantly inhibited the expression of the mesenchymal marker N-cadherin and upregulated the expression of the epithelial marker, E-cadherin. Compound 15k inhibited the expression of key proteins known to induce EMT (i.e., DVL3, ß-catenin, c-Myc) and upregulated the anti-metastatic protein, cyclin B1. Overall, in vitro, 15k significantly inhibited CRC progression and metastasis by inhibiting apoptosis, tubulin activity and the EMT pathways. Overall, these data suggest that compound 15k should be tested in vivo in a CRC animal model for further development.

HM015k, a Novel Silybin Derivative, Multi-Targets Metastatic Ovarian Cancer Cells and Is Safe in Zebrafish Toxicity Studies.

This study was designed to determine the in vitro mechanisms by which the novel silybin derivative, (E)-3-(3-(benzyloxy) phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (HM015k or 15k), produces its anticancer efficacy in ovarian cancer cells. Compound 15k induced apoptosis in ovarian cancer cells in a time-dependent manner by significantly upregulating the expression of Bax and Bak and downregulating the expression of Bcl-2. Interestingly, 15k induced the cleavage of Bax p21 into its more efficacious cleaved form, Bax p18. In addition, caspase 3 and caspase 9 were cleaved to their active forms, inducing the cleavage of poly ADP ribose polymerase (PARP) and ß-catenin. Furthermore, in OV2008 cells, 15k induced significant cleavage in nuclear ß-catenin to primarily inactive fragments of lower molecular weight. Furthermore, 15k reversed the metastatic potential of OV2008 cells by inhibiting their migration and invasiveness. The mesenchymal phenotype in OV2008 was reversed by 15k, causing cells to be rounder with epithelial-like phenotypes. The 15k-induced reversal was further confirmed by significant upregulation of the E-cadherin expression, an epithelial marker, while N-cadherin, a mesenchymal marker, was downregulated in OV2008 cells. Compound 15k inhibited the expression of the oncogenic c-Myc protein, downregulated proteins DVL3 and DVL2 and significantly upregulated cyclin B1. Also, 15k significantly downregulated the expression levels of ABCG2 and ABCB1 transporters in resistant ABCG2 overexpressing H460/MX20 and resistant ABCB1 overexpressing MDCK/MDR1 cells, respectively. Finally, 15k was safe in zebrafish in vivo model at concentrations up to 10 µM and induced no major toxicities in cardiac, morphology and swimming position parameters. Overall, 15k is a multi-targeted inhibitor with efficacy against metastatic and resistant ovarian cancer. Future in vivo studies will be conducted to determine the efficacy of 15k in tumor-bearing animals.