Vitamin D and Cellular Ca²⁺ Signaling in Breast Cancer

Apoptosis, Ca²⁺ Signaling and Vitamin D

Apoptosis in cancer. Apoptosis, a highly regulated form of cell death, is the main mechanism for controlling cell number in most tissues (1). Dysregulation of apoptosis underlies in the pathophysiology of proliferative disorders, and a decrease in apoptotic cell death contributes to cancer development and resistance to treatment (2). Moreover, conditions that inhibit or reduce apoptosis are associated with tumorigenesis and increased risk for several types of cancer in humans (1). Understanding how cell fate decisions are made and how cell death pathways are executed or held in check is pivotal to preventing and treating cancer. An induction of apoptosis is considered a potentially effective strategy for cancer treatment (1, 3). The main obstacle for the use of this approach is that the apoptotic molecular targets need to be selectively activated in cancer cells, and those targets need to be conclusively identified for different types of cancer.

Cellular Ca²⁺ and apoptosis. Cellular Ca²⁺ signals have been implicated in induction of apoptosis and regulation of the apoptotic pathways (2, 4-8). Ca²⁺ is considered the most versatile, ubiquitous intracellular messenger. It reversibly binds to specific proteins that act as Ca²⁺ sensors to decode information before passing it on to targets, but Ca²⁺ can also bind directly to target proteins. The membrane Ca²⁺ transport systems control cellular Ca²⁺ homeostasis. Ca²⁺ carries information to virtually all processes important for cell life, but it also transmits signals that promote cell death. Spatiotemporal characteristics of the Ca²⁺ signal determine the type and magnitude of biological responses, e.g. oscillations of cytosolic Ca²⁺ in pancreatic β-cells underlie the oscillatory pattern of insulin release from these cells (9, 10).

The exact mechanism of Ca²⁺ signaling in apoptosis is not fully understood. Our group (2, 8, 11-16) and others (4-6) have shown that increases in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) occur in the early and late stages of apoptosis. The critical characteristic of the apoptotic Ca²⁺ signal is a sustained increase in [Ca²⁺]_i, reaching elevated, but not cytotoxic levels. However, interactions of the cellular Ca²⁺ signal with molecular Ca²⁺ targets in cells undergoing apoptosis have not yet been conclusively identified. Ca²⁺-dependent intracellular cysteine proteases, the caspases (e.g. caspase-12), and Ca²⁺-dependent intracellular neutral proteases, the calpains, are considered as the primary Ca²⁺-activated apoptotic targets (2, 13, 17, 18).

Regulation of the Ca²⁺ signal via intracellular Ca²⁺ buffers plays a particularly important role in the apoptotic process. A key element of the cytosolic Ca²⁺ buffering system are the vitamin D-dependent Ca²⁺-binding proteins, calbindins. Our studies have shown (8, 13, 19, 20) that elevated levels of calbindin-D_28k dramatically increase the cytosolic Ca²⁺-buffering capacity and that an increase in Ca²⁺ buffering via forced expression of calbindin-D_28k protects cancer cells against Ca²⁺-mediated apoptosis. It is noteworthy that breast cancer cells do not express endogenous calbindin-D_28k and 1,25(OH)₂D₃ does not induce protein expression in these cells (13, 14).

We hypothesized (2, 7, 8) that a sustained increase in [Ca²⁺]_i, signals the cell to enter an apoptotic state via activation of the Ca²⁺-dependent protease μ-calpain followed by activation of the Ca²⁺/calpain-dependent caspase-12 and downstream caspases (e.g. caspase-3). A lack of expression or low levels of cytosolic Ca²⁺-binding proteins (e.g. calbindin-D_28k) may diminish the ability of the cell to buffer [Ca²⁺]_i increase and, thus, facilitate induction of apoptosis. On the other hand, agents that induce expression of the intracellular Ca²⁺ buffers (e.g. calbindin-D_28k by 1,25(OH)₂D₃ in certain normal cell types: intestine, kidney, mammary gland) or suppress pathways for the generation of the apoptotic Ca²⁺ signal (e.g. Ca²⁺ channel blockers) may protect against Ca²⁺-mediated apoptosis.

1,25(OH)₂D₃ and Ca²⁺-mediated apoptosis. It is well established that 1,25(OH)₂D₃ can induce Ca²⁺ signals in different cell types. 1,25(OH)₂D₃ activates voltage-dependent (VDCC) and voltage-insensitive (VICC) Ca²⁺ channels and triggers Ca²⁺ release from the endoplasmic reticulum (ER) through inositol 1,4,5-trisphosphate receptors and ryanodine receptors (2, 7, 8, 13, 14).

1,25(OH)₂D₃ generates biological responses via both genomic and nongenomic mechanisms (8, 21-24). Genomic responses utilize signal transduction pathways linked to the nuclear/cytosolic vitamin D receptors (VDRs), while nongenomic, rapid responses utilize signal transduction pathways coupled to the membrane VDRs. Analogs of vitamin D that can act as agonists and antagonists of these pathways have been identified (22, 24, 25). It appears that Ca²⁺ signals (transient and prolonged) triggered by 1,25(OH)₂D₃ can be linked to both membrane and nuclear VDRs (2, 8).

Our group (8, 11, 13, 14) and others (26, 27) have demonstrated that 1,25(OH)₂D₃ induces apoptosis in cancer cells and that apoptosis induced by 1,25(OH)₂D₃ depends on Ca²⁺ signaling (2, 10, 13, 28). Nuclear VDRs are believed to determine responsiveness of cancer cells to 1,25(OH)₂D₃. However, the efficacy of vitamin D analogs in cancer does not always correlate with their binding affinity to nuclear VDRs, and not all cancer cell lines expressing them respond to 1,25(OH)₂D₃. Therefore, signal transduction pathways coupled to both membrane and nuclear VDRs may be involved in regulation of apoptotsis (2, 8). Agonists of membrane VDRs can trigger the apoptotic Ca²⁺ signal and induce cell death without exerting the systemic calcemic activity of 1,25(OH)₂D₃ (13).

Below we summarize our findings regarding the role of 1,25(OH)₂D₃ in generating Ca²⁺ signals in breast cancer cells and provide evidence that 1,25(OH)₂D₃-induced Ca²⁺ signals can determine fate of these cells by apoptosis. These findings may help in the rational search for therapeutic and preventive agents for breast cancer that act via Ca²⁺-dependent molecular targets in apoptotic pathways.

1,25(OH)₂D₃-induced Ca²⁺ Signaling and Apoptosis in Breast Cancer Cells

Intracellular Ca²⁺, 1,25(OH)₂D₃ and apoptosis in human breast cancer cells. Our early findings indicate that the plasma membrane VICC and the ER Ca²⁺ release channels are the main pathways for Ca²⁺ entry and Ca²⁺ mobilization in breast cancer cells and that 1,25(OH)₂D₃ increases Ca²⁺ influx through VICC and depletes the ER Ca²⁺ stores in these cells (10, 14, 15). We suggested that targeting of Ca²⁺ signaling mediated by VICC and the ER Ca²⁺ may stand as a novel approach to the treatment and prevention of breast cancer.

We have characterized in detail the regulation of intracellular Ca²⁺ in the estrogen receptor-positive human breast cell line MCF-7 (10-12). These cells express the highly permeable VICC, but not VDCC. The ER is a major Ca²⁺ storage compartment, and mobilization of Ca²⁺ from the ER occurs through inositol 1,4,5-trisphosphate receptor/Ca²⁺ release channel, while the ryanodine receptor/Ca²⁺ release channel is not expressed. 1,25(OH)₂D₃ rapidly increases Ca²⁺ influx through VICC, depletes the ER Ca²⁺ stores and elevates basal [Ca²⁺]_i. 1,25(OH)₂D₃-evoked increase in [Ca²⁺]_i is associated with induction of apoptosis in MCF-7 cells (as evaluated by DNA fragmentation and morphological criteria). Treatment of these cells with a Ca²⁺ ionophore, ionomycin, similarly induces apoptosis. MCF-7 cells loaded with the cytosolic Ca²⁺ buffer (BAPTA) do not undergo apoptosis in response to 1,25(OH)₂D₃. These findings imply that 1,25(OH)₂D₃ triggers apoptosis in breast cancer cells by causing an increase in Ca²⁺ entry through VICC and depletion of the ER Ca²⁺ stores. The resulting elevated [Ca²⁺]_i appears to be sufficient to elicit apoptosis.

Ca²⁺ and calpain as mediators of apoptosis in breast cancer cells. In a specific study (13), the mechanism of Ca²⁺-mediated apoptosis in breast cancer cells was investigated. An increase in [Ca²⁺]_i and depletion of the ER Ca²⁺ stores with 1,25(OH)₂D₃ induced apoptosis in MCF-7 cells. The increase in [Ca²⁺]_i was associated with activation of the Ca²⁺-dependent cysteine protease, μ-calpain. The forced expression of the Ca²⁺-binding protein calbindin-D_28k in MCF-7 cells not only attenuated the elevation in [Ca²⁺]_i and μ-calpain activation, but also reduced apoptotic death triggered by 1,25(OH)₂D₃. Similarly, the inhibition of calpain activity by structurally unrelated inhibitors reduced the proportion of apoptotic cells. These results indicate that calpain may play the role of the major protease in apoptotic cell death.

1,25(OH)₂D₃ induces Ca²⁺-mediated apoptosis in breast cancer cells, but not normal cells. Another study aimed in comparing the effects of 1,25(OH)₂D₃ on Ca²⁺ signaling and apoptosis in the cancer and normal human mammary epithelial cells (HMECs) (8). The treatment of MCF-7 breast cancer cells with 1,25(OH)₂D₃ induced a sustained increase in [Ca²⁺]_i and activated Ca²⁺-dependent apoptotic proteases, μ-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the calpain peptide substrate. The selective inhibition of the Ca²⁺-binding sites of μ-calpain reduced apoptotic indices in 1,25(OH)₂D₃-treated cells. 1,25(OH)₂D₃ did not induce apoptosis in normal HMECs, as evaluated by DNA fragmentation, loss of plasma membrane asymmetry and morphological criteria. In HMECs, 1,25(OH)₂D₃ triggered a transient Ca²⁺ response, which was not accompanied by calpain or caspase activation. HMECs, but not MCF-7 cells, expressed the Ca²⁺-binding protein calbindin-D_28k and were capable of buffering the apoptotic (i.e. non-cytotoxic) [Ca²⁺]_i increases induced by the Ca²⁺ ionophore ionomycin.

These results support the hypothesis that the Ca²⁺ entry, mobilization, and -buffering mechanisms differ dramatically in breast cancer cells and normal mammary epithelial cells. Ca²⁺ handling by normal HMECs (Ca²⁺ entry and Ca²⁺ mobilization pathways allowing only a transient 1,25(OH)₂D₃-induced Ca²⁺ increase and a large Ca²⁺-buffering capacity) seem sufficient to protect those cells from Ca²⁺-mediated apoptosis. Ca²⁺ handling by breast cancer cells (Ca²⁺ entry and Ca²⁺ mobilization pathways permitting generation of a sustained, prolonged increase of [Ca²⁺]_i and a low Ca²⁺-buffering capacity) allows the induction of apoptosis with 1,25(OH)₂D₃ in these cells. The findings clearly imply that differences of Ca²⁺ regulatory mechanisms in breast cancer cells vs. normal mammary epithelial cells underlie resistance of normal cells and susceptibility of cancer cells to 1,25(OH)₂D₃-induced, Ca²⁺-mediated apoptosis.