The purpose of this study is to demonstrate MCF-7 cells' dependence on calcium for growth and to exploit that dependence to improve chemotherapy efficacy.Fura-2 fluorescence imaging shows that MCF-7 cells maintain a higher basal intracellular calcium concentration than non-tumorigenic MCF-10A cells.Blocking T-type calcium channels with mibefradil reduced MCF-7 intracellular calcium concentration.Flow cytometry shows that knocking down T-type calcium channel expression with siRNA caused an increase in MCF-7 cells in G1 phase and a decrease in cells in S phase.Proliferation assays of MCF-7 cells treated with EGTA and thapsigargin reveal the dependence of MCF-7 cell growth on extracellular and intracellular calcium sources, respectively.In vitro, interlaced treatment that alternated the T-type calcium channel blocker NNC-55-0396 with paclitaxel more effectively reduced MCF-7 cell number than chemotherapy alone.In a mouse in vivo model, interlaced mibefradil and paclitaxel more effectively reduced MCF-7 xenograft size than chemotherapy alone.These findings indicate that MCF-7 cells are dependent on calcium for proliferation, particularly in passing the G1/S cell cycle checkpoint.Further, this dependence on calcium can be exploited by alternating treatment with T-type calcium channel blockers with paclitaxel in an interlaced therapy scheme that increases the efficacy of the chemotherapy.

Abstract T-type Ca2+ channel mediated Ca2+ influx is important for tumor cell proliferation. In order to understand the role of this influx in tumor cell cycling, we conducted experiments to block T-type calcium channels in MCF7 breast cancer cells using pharmacological inhibitors, mibefradil or its more T-type calcium channel specific derivative NNC-55-0396, and examined the effects of this blockade on the passage through the cell cycle of MCF7 cells and whether T-type calcium channel inhibition can be used in conjunction with paclitaxel chemotherapy to increase the efficacy of the chemotherapy. Flow cytometry shows that blocking T-type calcium channels with the specific inhibitor NNC-55-0396 resulted in an increase in MCF7 cells in the G1 phase and a decrease in cells in the S phase of the cell cycle. Treatment with paclitaxel for 72 hours in vitro yielded lower MCF7 cell numbers than treatment with NNC-55-0396, as measured by a cell proliferation assay. However, a treatment regimen alternating NNC-55-0396 with paclitaxel over 72 hours was found to reduce MCF7 cell number to a larger degree than either agent alone for the same time period. For in vivo modeling, MCF7 xenografts were implanted in the mammary fat pads of nude mice, with tumor size monitored via caliper measurements. Similar to the in vitro findings, a treatment regiment that alternated paclitaxel chemotherapy with the calcium channel inhibitor mibefradil reduced tumor size to a larger degree than either agent alone, though that difference did not rise to a statistically significant level. These results indicate that blocking T-type calcium channels halts proliferating MCF7 cells at the G1/S checkpoint, and that this phenomenon may lead to an increased sensitivity to paclitaxel chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5672. doi:1538-7445.AM2012-5672

We have measured the expression of T-type Ca2+ channel mRNA in breast cancer cell lines (MCF-7 (ERα+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both α1G and α1H isoforms of T-type Ca2+ channels. In order to further clarify the role of T-type Ca2+ channels in proliferation, we tested the effects of a selective T-type Ca2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERα+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca2+ channels with siRNA targeting both α1G and α1H resulted in growth inhibition as much as 45% ± 5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.

Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells, free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear; however, there is a growing body of evidence that suggests the T-type Ca(2+) channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca(2+) channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel is minimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca(2+) channels are not expressed in epithelial cells, selective T-type Ca(2+) channel blockers may be useful in the treatment of certain types of cancers.

The efficacy of the biphasic poroviscoelastic (BPVE) theory [1] in constitutive modeling of articular cartilage biomechanics is well-established [2–4]. Indeed, this model has been used to simultaneously predict stress relaxation force across confined compression, unconfined compression, and indentation protocols [2,3]. Previous works have also demonstrated success in simultaneously curve-fitting the BPVE model to reaction force and lateral deformation data gathered from stress relaxation tests of articular cartilage in unconfined compression [4]. However, a potential limitation of practical applications of such a successful model is seen in some commonly-employed mechanical testing methods for articular cartilage, such as confined compression and unconfined compression. These methods require the excision of a disk of cartilage from its underlying subchondral base, which likely would compromise the structural integrity of the tissue, causing swelling and curling artifacts of the sample [5]. Indentation represents a testing protocol that can be used with an intact cartilage layer. This results in a specimen more closely resembling cartilage in vivo. Using an agarose gel construct, our previous study [6] has demonstrated that a unique set of the six BPVE model parameters of a soft tissue can be determined readily from in situ dual indentation method using stress relaxation and creep viscoelastic protocols. The objective of the current study is to validate the efficacy of this technique as a means to determine the BPVE material parameters of articular cartilage.