Potent Killing of Paclitaxel- and Doxorubicin-resistant Breast Cancer Cells by Calphostin C Accompanied by Cytoplasmic Vacuolization

Abstract

Drug resistance is a major impediment to the successful treatment of breast cancer using chemotherapy. The photoactivatable drug calphostin C has shown promise in killing select drug-resistant tumor cells lines in vitro. To assess the effectiveness of this agent in killing doxorubicin- or paclitaxel-resistant breast tumor cells and to explore its mode of action, MCF-7 cells were exposed to increasing concentrations of either doxorubicin or paclitaxel until maximum resistance was obtained. This resulted in the creation of isogenic drug-resistant MCF-7TAX and MCF-7DOX cell lines, which were approximately 50- and 65-fold resistant to paclitaxel and doxorubicin, respectively. Interestingly, calphostin C was able to kill MCF-7TAX cells as efficiently as wildtype MCF-7 cells (IC50s were 9.2 and 13.2 nM, respectively), while MCF-7DOX cells required a 5-fold higher concentration of calphostin C to achieve the same killing (IC50 = 64.2 nM). Consistent with their known mechanisms of action, paclitaxel killed tumor cells by inducing mitotic arrest and cell multinucleation, while doxorubicin induced plasma membrane blebbing and decreased nuclear staining with propidium iodide. In contrast, cytoplasmic vacuolization accompanied cell killing by calphostin C in these cell lines, without the induction of caspase-8 or PARP cleavage or the release of cytochrome c from mitochondria. Calphostin C had little effect on the uptake of either paclitaxel or doxorubicin by the cells. Taken together, the above data suggests that calphostin C is able to potently kill drug-resistant breast tumor cells through a mechanism that may involve the induction of cytoplasmic vacuolization, without activation of typical apoptotic pathways. Consequently, calphostin C may prove useful clinically to combat tumor growth in breast cancer patients whose tumors have become unresponsive to anthracyclines or taxanes, particularly in association with photodynamic therapy.

Non-apoptotic programmed cell death induced by a copper(II) complex in human fibrosarcoma cells

“…On the contrary, no evidence of either feature has been detected in the A 0 -treated cells, thus excluding that the copper complex triggers an apoptotic pathway. Indeed, while selected examples of caspase-independent or nuclear fragmentation-independent apoptotic deaths have been described (see for review, respectively, Kolenko et al 2000;and Ferri and Kroemer 2000), the simultaneous absence of both features is considered a hallmark of nonapoptotic cell death (Szende et al 1995;Sasaki et al 2003;Guo et al 2003;Wojcik et al 2004;Kelly et al 2004;Wang et al 2004;Hirai and Harada 2004;Naumann et al 2004). The results presented also demonstrate that A 0 inhibits the activation of caspase-3 caused by cisplatin.…”

Tamoxifen Inhibits TRPV6 Activity via Estrogen Receptor–Independent Pathways in TRPV6-Expressing MCF-7 Breast Cancer Cells

“…When the PKC inhibitor calphostin C (100 nmol/L) was applied, TRPV6 activity was diminished. Furthermore, tamoxifen had no inhibitory effect in the presence of calphostin C. It was shown previously that calphostin C can reduce MCF-7 cell viability very potently (43). However, because TRPV6-mediated barium entry was also inhibited by tamoxifen, and because barium does not bind to calmodulin (44), we conclude that tamoxifen does not inhibit TRPV6 function through augmentation of calmodulin-mediated inactivation.…”

Cytoplasmic vacuolization during exposure to drugs and other substances

“…Compartments on autophagy to lysosome pathway are vacuolated upon exposure to various substances including chloroquine (Fedorko et al 1968). Endoplasmic reticulum is vacuolated by a panel of substances such as taxol (Chen et al 2008), calphostin C (Guo et al 2003), and 15-deoxy-Δ 12,14 -prostaglandin J 2 (Kar et al 2009) been shown that organic cation transporter (OCT), a member of the SLC family, plays major role in the delivery of organic amines into cells (Koepsell et al 2007). Carrier-mediated processes would be expected to play important roles in the transport of organic amines into the cells when these agents are added at concentrations around and below the K m value of the carrier systems.…”

“…Taxol, an anticancer drug, also has been shown to cause ER-derived vacuolization when added at relatively high concentrations (e.g., 70 μM) compared with the dose used to induce mitotic arrest (less than 100 nM) (Chen et al 2008). Interestingly, calphostin C, a well-known inhibitor for protein kinase C, induces ER-derived vacuolation and subsequent death of the cells that are resistant to pacilitaxel (taxol) (Guo et al 2003;Kaul and Maltese 2009), indicating its possible usefulness for cancer therapy. Cytoplasmic vacuolization derived from enlarged/dilated ER upon exposure to these drugs could be rationally interpreted as a consequence of excessive ER stress.…”