Apoptosis Inducing Activity of Rhinacanthin-C in Doxorubicin-Resistant Breast Cancer MCF-7 Cells.

Rhinacanthin-C is a natural bioactive naphthoquinone ester with potential chemotherapeutic value in cancer treatment. In this study, we investigated its apoptotic induction ability and the involved mechanisms through the mitogen-activated protein kinases (MAPK) and protein kinase B/glycogen synthase kinase-3ß/nuclear factor erythroid 2-related factor 2 (Akt/GSK-3ß/Nrf2) signaling pathways in doxorubicin-resistant breast cancer MCF-7 (MCF-7/DOX) cells. Our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that rhinacanthin-C (3-28 µM) significantly decreased the viability of MCF-7/DOX cells and potentiated hydrogen peroxide cytotoxicity. This naphthoquinone was able to increase intracellular reactive oxygen species (ROS), as measured by the 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. This compound increased the number of apoptotic cells by elevating the ratio of apoptotic checkpoint proteins Bax/Bcl-2 and by decreasing the expression of poly(ADP-ribose) polymerase (PARP) protein. Furthermore, Western blotting analyses showed that treatment with rhinacanthin-C (3-28 µM) for 24 h significantly decreased the expression levels of the phosphorylated forms of MAPK proteins (i.e., extracellular signal regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38), Akt, GSK-3ß and Nrf2 proteins in MCF-7/DOX cells. Inhibition of the Akt/GSK-3ß/Nrf2 pathway led to a significant reduction in heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NADP)(H): quinone oxidoreductase 1 (NQO1) proteins. These findings suggested that rhinacanthin-C was able to induce apoptosis in MCF-7/DOX cells through increased ROS production and suppression of the cell survival systems mediated by the MAPKs and Akt/GSK-3ß/Nrf2 signaling pathways.

Synthesis and antitumor activity of novel pyridinium fullerene derivatives.

PURPOSE: We have previously reported that some cationic fullerene derivatives exhibited anticancer activity, and they are expected to be a potential lead compound for an anti-drug resistant cancer agent. However, they are bis-adducts and a mixture of multiple regioisomers, which cannot be readily separated due to the variability of substituent positions on the fullerene cage. To overcome this issue, we evaluated the antiproliferative activities of a set of mono-adduct derivatives and examined their structure-activity relationship. In addition, the in vivo antitumor activity of selected derivatives was also examined. METHODS: Nineteen pyridinium fullerene derivatives were newly designed and synthesized in this study. Their antiproliferative activities were evaluated using several cancer cell lines including drug-resistant cells. Furthermore, in vivo antitumor activity of several derivatives was investigated in mouse xenograft model of human lung cancer. RESULTS: The derivatives inhibited the proliferation of cancer cell lines, including cisplatin-resistant cells and doxorubicin-resistant cells. It was also shown that compound 10 (10 µM), 13 (10 µM) and cis-14 (10 µM) induced the intracellular oxidative stress. In addition, compound 13 (20 mg/kg) and cis-14 (15 mg/kg) significantly exhibited antitumor activity in mouse xenograft model of human lung cancer. CONCLUSION: We synthesized a novel set of mono-adduct fullerene derivatives functionalized with pyridinium groups and found that most of them show potent antiproliferative activities against cancer cell lines and some of them show significant antitumor activities in vivo. We propose that these fullerene derivatives serve as the lead compounds for a novel type of antitumor agents.

Transferrin-Conjugated Polymeric Nanoparticle for Receptor-Mediated Delivery of Doxorubicin in Doxorubicin-Resistant Breast Cancer Cells.

In this study, a transferrin (Tf)-conjugated polymeric nanoparticle was developed for the targeted delivery of the chemotherapeutic agent doxorubicin (Dox) in order to overcome multi-drug resistance in cancer treatment. Our objective was to improve Dox delivery for producing significant antitumor efficacy in Dox-resistant (R) breast cancer cell lines with minimum toxicity to healthy cells. The results of our experiments revealed that Dox was successfully loaded inside a transferrin (Tf)-conjugated polymeric nanoparticle composed of poloxamer 407 (F127) and 123 (P123) (Dox/F127&P123-Tf), which produced nanosized particles (~90 nm) with a low polydispersity index (~0.23). The accelerated and controlled release profiles of Dox from the nanoparticles were characterized in acidic and physiological pH and Dox/F127&P123-Tf enhanced Dox cytotoxicity in OVCAR-3, MDA-MB-231, and MDA-MB-231(R) cell lines through induction of cellular apoptosis. Moreover, Dox/F127&P123-Tf inhibited cell migration and altered the cell cycle patterns of different cancer cells. In vivo study in MDA-MB-231(R) tumor-bearing mice demonstrated enhanced delivery of nanoparticles to the tumor site when coated in a targeting moiety. Therefore, Dox/F127&P123-Tf has been tailored, using the principles of nanotherapeutics, to overcome drug-resistant chemotherapy.

Experimental therapy of doxorubicin resistant human uveal melanoma with targeted cytotoxic luteinizing hormone-releasing hormone analog (AN-152).

BACKGROUND: Cytotoxic analogs of LHRH (luteinizing hormone-releasing hormone) can be successfully used for the treatment of hormone-dependent cancers such as prostatic, ovarian, endometrial, but our knowledge about their effect on hormone-independent cancers such as human uveal melanoma (UM) is limited. Previously, we have demonstrated that 46% of UM express full-length LHRH receptors. This finding has led us to further examine the mechanism of action of LHRH receptor based targeted therapies in this malignancy. AIMS: In the present study we investigated the cellular uptake of doxorubicin (DOX) and cytotoxic LHRH analog AN-152 (AEZS-108, zoptarelin doxorubicin) on human UM cell lines (OCM3) and its DOX resistant form OCM3DOX320 by confocal laser scanning microscopy. The LHRH receptor expression was characterized by RT-PCR and immunocytochemistry. RESULTS: We were able to establish a new, stable and DOX resistant human UM cell line OCM3DOX320. Our results demonstrated the expression of splice variants and isoforms of receptor for LHRH in OCM3 UM cell line and its doxorubicin resistant form OCM3DOX320. It has been revealed by MTT assay that AN-152 inhibited cell proliferation in a dose dependent manner in OCM3DOX320 cells. Furthermore, receptor-mediated uptake of AN-152 was demonstrated using confocal laser scanning microscopy in both cell line. CONCLUSIONS: Our results suggest that the antiproliferative effect of AN-152 can be detected even if only LHRH receptor isoforms are expressed. Our study also demonstrates the LHRH receptor-mediated uptake of AN-152 in DOX resistant OCM3DOX320 cells. Our experiments provide new insights into a potential targeted therapy of UM and give further details about the accumulation of AN-152 in hormone-independent DOX-resistant cells expressing splice variants of the LHRH receptors.

Increased lipid peroxidation, apoptosis and selective cytotoxicity in colon cancer cell line LoVo and its doxorubicin-resistant subline LoVo/Dx in the presence of newly synthesized phenothiazine derivatives.

Cancer cells often develop the resistance to pro-apoptotic signaling that makes them invulnerable to conventional treatment. Therapeutic strategies that make cancer cells enter the path of apoptosis are desirable due to the avoidance of inflammatory reaction that usually accompanies necrosis. In the present study phenothiazines (fluphenazine and four recently synthesized derivatives) were investigated in order to identify compounds with a potent anticancer activity. Since phenothiazines are known as multidrug resistance modulators the sensitive human colorectal adenocarcinoma cell line (LoVo) and its doxorubicin-resistant, ABCB1 overexpressing, subline (LoVo/Dx) have been employed as a model system. In studied cancer cells cytotoxic effect of the phenothiazine derivatives was accompanied by apoptosis and autophagy induction as well as by the increase of cellular lipid peroxidation and intracellular reactive oxygen species generation. Molecular modelling revealed that reactivity of phenothazines (manifested by their low energy gap) but not lipophilicity was positively correlated with their anticancer potency, pro-oxidant properties and apoptosis induction ability. Additionally, some of the studied compounds turned out to be more potent cytotoxic and pro-apoptotic agents in doxorubicin-resistant (LoVo/Dx) cells than in sensitive ones (LoVo). The hypothesis was assumed that studied phenothiazine derivatives induced apoptotic cell death by increasing the production of reactive oxygen species.