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.

Novel Curcumin-Resveratrol Solid Nanoparticles Synergistically Inhibit Proliferation of Melanoma Cells.

Polyphenols such as curcumin (Cur) and resveratrol (Res) have been recently shown to have potential to inhibit proliferation of highly aggressive melanoma cells. This study was designed to investigate the feasibility of a topical delivery system, using a solid lipid nanoparticles (SLNs) loaded delivery systems, that can enhance the skin penetration and anti-cancer efficacy of combination of these polyphenols. Negatively charged Cur-Res SLNs with a mean diameter of 180.2 ± 7.7 nm were prepared using high shear homogenization method. Cur-Res SLNs were found to be stable up to 2 weeks under 4°C. The in vitro release study showed that Res was released five time more than curcumin. The permeability of resveratrol was about 1.67 times that of curcumin from the SLN-gel formulation which was significantly (p < 0.05) lower than from SLN suspension. More than 70% of Cur-Res SLNs were bound to skin locally in a skin binding study suggesting potentially utility of Cur-Res SLNs in the treatment of localized melanoma. In fact, the electrical cell-substrate impedance sensing (ECIS) measurements suggested that Cur-Res combination has potential to stop cell migration of B16F10 melanoma cells. Furthermore, both, Cur-Res SLNs and Cur-Res solution at the ratio of 3:1 demonstrated a strong synergistic inhibition of SK-MEL-28 melanoma cell proliferation. Further evaluation of Cur-Res SLNs in vivo melanoma models are warranted to establish the clinical utility of Cur-Res formulations in melanoma therapy.

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.

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.

lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth of colon cancer cells via apoptosis and sub G1 cell cycle arrest.

A series of lH-pyrazolo[3,4-b]quinolin-3-amine derivatives were synthesized and evaluated for anticancer efficacy in a panel of ten cancer cell lines, including breast (MDAMB-231 and MCF-7), colon (HCT-116, HCT-15, HT-29 and LOVO), prostate (DU-145 and PC3), brain (LN-229), ovarian (A2780), and human embryonic kidney (HEK293) cells, a non-cancerous cell line. Among the eight derivatives screened, compound QTZ05 had the most potent and selective antitumor efficacy in the four colon cancer cell lines, with IC50 values ranging from 2.3 to 10.2 µM. Furthermore, QTZ05 inhibited colony formation in HCT-116 cells in a concentration-dependent manner. Cell cycle analysis data indicated that QTZ05 caused an arrest in the sub G1 cell cycle in HCT-116 cells. QTZ05 induced apoptosis in HCT-116 cells in a concentration-dependent manner that was characterized by chromatin condensation and increase in the fluorescence of fluorochrome-conjugated Annexin V. The findings from our study suggest that QTZ05 may be a valuable prototype for the development of chemotherapeutics targeting apoptotic pathways in colorectal cancer cells.

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.

Novel Thienopyrimidine Derivative, RP-010, Induces ß-Catenin Fragmentation and Is Efficacious against Prostate Cancer Cells.

Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. The compound RP-010 was efficacious against both PC-3 and DU145 prostate cancer (PC) cells (IC50 < 1 µM). The cytotoxicity of RP-010 was significantly lower in non-PC, CHO, and CRL-1459 cell lines. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in G2 phase of the cell cycle, and induced mitotic catastrophe and apoptosis in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) affected the wingless-type MMTV (Wnt)/ß-catenin signaling pathway, in association with ß-catenin fragmentation, while also downregulating important proteins in the pathway, including LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the Wnt pathway. In addition, RP-010 (0.5, 1, 2 and 4 µM) significantly decreased the migration of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations of up to 6 µM. In conclusion, RP-010 may be an efficacious and relatively nontoxic anticancer compound for prostate cancer. Future mechanistic and in vivo efficacy studies are needed to optimize the hit compound RP-010 for lead optimization and clinical use.

Cariprazine, A Dopamine D2/D3 Receptor Partial Agonist, Modulates ABCG2-Mediated Multidrug Resistance in Cancer.

Multidrug resistance (MDR) is a continuing clinical problem that limits the efficacy of chemotherapy in cancer. The over expression of the ATP-binding cassette (ABC) family G2 (ABCG2) transporter is one of the main mechanisms that mediates MDR in cancer. Molecular modeling data indicated that cariprazine, a dopamine D2/D3 receptor partial agonist, had a significant binding affinity for ABCG2 transporter with a Glide XP score of -6.515. Therefore, in this in vitro study, we determined the effect of cariprazine on MDR resulting from the overexpression of ABCG2 transporters. Alone, cariprazine, at concentrations up to 20 µM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 10 µM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1⁻20 µM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2-overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. Cariprazine, in a concentration-dependent (1⁻20 µM), significantly increased the intracellular accumulation of Rhodamine 123 in S1M1-80. Interestingly, 10 or 20 µM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and expression of ABCG2 transporters at nontoxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can resensitize resistant cancer cells to mitoxantrone.

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.

Design and discovery of silybin analogues as antiproliferative compounds using a ring disjunctive - Based, natural product lead optimization approach.

The present study reports the synthesis and anticancer activity evaluation of twelve novel silybin analogues designed using a ring disjunctive-based natural product lead (RDNPL) optimization approach. All twelve compounds were tested against a panel of cancer cells (i.e. breast, prostate, pancreatic, and ovarian) and compared with normal cells. While all of the compounds had significantly greater efficacy than silybin, derivative 15k was found to be highly potent (IC50 < 1 µM) and selective against ovarian cancer cell lines, as well as other cancer cell lines, compared to normal cells. Preliminary mechanistic studies indicated that the antiproliferative efficacy of 15k was mediated by its induction of apoptosis, loss of mitochondrial membrane potential and cell cycle arrest at the sub-G1 phase. Furthermore, 15k inhibited cellular microtubules dynamic and assembly by binding to tubulin and inhibiting its expression and function. Overall, the results of the study establish 15k as a novel tubulin inhibitor with significant activity against ovarian cancer cells.

The dopamine D3 receptor antagonists PG01037, NGB2904, SB277011A, and U99194 reverse ABCG2 transporter-mediated drug resistance in cancer cell lines.

The ATP - binding cassette (ABC) family G2 (ABCG2) transporters are known to produce multidrug resistance (MDR) in cancer, thereby limiting the clinical response to chemotherapy. Molecular modeling data indicated that certain dopamine (DA) D3 receptor antagonists had a significant binding affinity for ABCG2 transporter. Therefore, in this in vitro study, we determined the effect of the D3 receptor antagonists PG01037, NGB2904, SB277011A, and U99194 on MDR resulting from the overexpression of ABCG2 transporters. The D3 receptor antagonists, at concentrations >100 µM, did not significantly affect the viability of H460-MX20, S1M1-80, A549-MX10 or wild type ABCG2 overexpressing (HEK293-R2) cells. However, at concentrations ranging from 0.01 to 10 µM, the D3 receptor antagonists PG01037, NGB2904, SB-277011A, and U99194 significantly increased the efficacy of the anticancer drugs mitoxantrone and doxorubicin in ABCG2-overexpressing MDR cells. Efflux studies indicated that both PG01037 and NGB2904, at a concentration of 5 µM, significantly decreased the efflux of rhodamine 123 from H460-MX20 cells. Interestingly, 5 µM of PG01037 or NGB2904 significantly decreased the expression levels of the ABCG2 protein, suggesting that these compounds inhibit both the function and expression of ABCG2 transporters at non-toxic concentrations.

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.

Thienopyrimidine derivatives exert their anticancer efficacy via apoptosis induction, oxidative stress and mitotic catastrophe.

In this study, a series of 13 structural variants of thieno[2,3d]pyrimidine derivatives (6a-6m) were synthesized and screened for cytotoxicity in a panel of colorectal, ovarian, and brain cancer cell lines. The selectivity of the compounds was assessed by determining the cytotoxicity in normal epithelial cell line (CHO). The most potent compound, 6j, was efficacious (with IC50 range of 0.6-1.2 µM) in colon (HCT116 and HCT15), brain (LN-229 and GBM-10) and ovarian (A2780 and OV2008) cancer cell lines. In contrast, in the normal cell line (CHO), the IC50 values for 6j were 14 ± 1.3 µM. Compound 6j significantly inhibited the clonogenic potential of HCT116, OV2008 and A2780 cell lines in concentration - dependent (0.5-4 µM) manner. Also, 6j induced 1) formation of reactive oxygen species; 2) apoptosis and 3) mitotic catastrophe in HCT116 and OV2008 cells (IC50 = 0.5-2 µM). Furthermore, apoptosis was the predominant mechanism of death in A2780 cells. The cytotoxicity of 6j in wild type HCT116 cells was similar to that in HCT116 cells lacking the apoptotic genes for Bax, Bak, or Bak and Bax, indicating that 6j induces mitotic catastrophe as alternative mechanism of death when when certain apoptotic proteins are absent. In summary, this study has identified a lead molecule, 6j, that selectively induces oxidative stress, apoptosis and mitotic catastrophe in specific cancer (colon and ovarian) cell lines.