Differential role of apoptosis and autophagy associated with anticancer effect of lupulone (hop ß-acid) derivatives on prostate cancer cells.

Lupulone, a ß-acid derived from hop extracts has been shown to exhibit cytotoxic activity against cancer cells. In this study we investigated the functional role of different modes of cell death that mediate anticancer effect of lupulone derivatives in prostate cancer cells. ELISA, immunoblotting and siRNA approaches were utilised to study cell death, expression of proteins of interest and their functional activities. We found that the anticancer effect of lupulone derivatives on prostate cancer cells is associated with induction of apoptosis and autophagy as determined by increases of DNA fragmentation and LC3I/ LC3II conversion respectively. Inhibition of apoptosis using a pan-caspase inhibitor resulted in increased levels of autophagy. Following screening of proteins associated with autophagy we found that Atg4ß expression was increased in prostate cancer cells after treatment with lupulone. Transfection of cells with siRNA against Atg4ß resulted in increased levels of apoptosis in prostate cancer cells. Treatment of prostate cancer cells with lupulone derivatives initiated two modes of cell death: apoptosis as a killing pathway and autophagy as a protection against cell death. Further studies are required to investigate the regulation of Atg4ß activity in lupulone derivatives-induced negative crosstalk between apoptosis and autophagy.

An investigation into the anticancer effects and mechanism of action of hop ß-acid lupulone and its natural and synthetic derivatives in prostate cancer cells.

Lupulone, a ß-acid derived from hop extracts has been shown to exhibit antibacterial and anticancer activity. In this study we investigated the anticancer potency of lupulone and its novel derivatives and their mechanism of action on prostate cancer cells. Cell viability was determined using the MTT assay, and the ELISA approach was used to investigate induction of apoptosis. Immunoblot analysis was carried out to determine activation and regulation of proteins associated with cell death. Screening of natural and new lupulone derivatives for their anticancer activity demonstrated that one (lupulone derivative 1h) displayed stronger anticancer activity than lupulone itself on PC3 and DU145 prostate cancer cells. We further found that lupulone derivatives induced caspase-dependent apoptosis that is associated with activation of caspases 8, 9, and 3. Furthermore, caspase 8 inhibitor Z-IETD-fmk reduced cell death induced by lupulone derivatives, suggesting that apoptosis is mediated by caspase 8. Finally, we found that lupulone and its synthetic derivatives also increased formation of LC3II suggesting that autophagy is also implicated in prostate cancer cell death. The new lupulone derivatives induce caspase-dependent apoptosis and autophagy in prostate cancer cells and appear to be good candidates for further preclinical studies of prostate cancer treatment.

Antiproliferative effects of CC-8062 and CC-8075 in pancreatic cancer cells.

OBJECTIVES: Pancreatic cancer is one of the leading causes of cancer related deaths in the western world. It is also resistant to most chemotherapeutic modalities. Phosphodiesterase-4 inhibitors (PDE4is) have found applications in the treatment of respiratory diseases. The aim of this study is to investigate the cytotoxic effect of 2 novel PDE4is, the CC-8075 and CC-8062 compounds in pancreatic cancer cells. METHODS: Cell proliferation was measured using the sulforhodamine B protein dye. Induction of apoptosis was detected using enzyme-linked immunosorbent assay. Regulation of proteins and posttranslational modifications were determined using immunoblotting. RESULTS: Treatment of pancreatic cancer cells with CC-8075 and CC-8062 reduces their proliferation and increases apoptosis that is caspase dependent in T3M4 cells. Furthermore, PDE4is increase phosphorylation of p38MAPK, mitogen-activated protein kinase (MAPK) kinase 3/6,MAPKYactivated protein kinase 2, Atf2, and Hsp27. The use of thep38MAPK-specific inhibitors SB202190 and SB203580 results in a modest reduction in PDE4i-induced apoptosis in T3M4 cells. Also, retinoids enhance apoptosis induced by CC-8075 and CC-8062 in GER cells. CONCLUSIONS: These results highlight the antiproliferative effects of the phosphodiesterase inhibitors CC-8075 and CC-8062 in pancreatic cancer cells and suggest that activation of p38MAPK signaling pathway may be associated with this process.

Inhibition of osteoblast function in vitro by aminobisphosphonates.

Bisphosphonates are analogues of pyrophosphate, a key physicochemical inhibitor of mineralisation. We examined the direct actions of bisphosphonates on the function of cultured osteoblasts derived from rat calvariae. Treatment with zoledronate, the most potent bisphosphonate studied, reduced osteoblast number at concentrations > or = 100 nM and was strongly toxic at 10 microM, causing a threefold decrease in osteoblast viability after 2 days and a 90% decrease in cell numbers after 14 days. In control osteoblast cultures on plastic, abundant formation of 'trabecular' mineralised bone matrix nodules began after 10 days. Continuous exposure to zoledronate inhibited bone mineralisation at concentrations as low as 10 nM. Pamidronate and clodronate exerted similar effects but at higher doses > or = 1 and > or = 10 microM, respectively). Short-term or intermittent exposure of osteoblasts to zoledronate and pamidronate (1-10 microM) was sufficient to inhibit bone mineralisation by > or = 85%. Zoledronate but not pamidronate or clodronate also strongly inhibited osteoblast alkaline phosphatase activity at concentrations > or = 100 nM and soluble collagen production at concentrations > or = 1 microM. We additionally studied the effects of zoledronate on osteoblasts cultured on dentine, a bone-like mineralised substrate, observing similar inhibitory effects, although at concentrations 10-100-fold higher; this shift presumably reflected adsorption of zoledronate to dentine mineral. Thus, zoledronate blocked bone formation in two ways: first, a relatively non-toxic, selective inhibition of mineralisation at concentrations in the low nanomolar range and second, a cytotoxic inhibition of osteoblast growth and function at concentrations > or = 1 microM. Although no data are available on the bisphosphonate concentrations that osteoblasts could be exposed to in vivo, our results are consistent with earlier observations that bisphosphonates may inhibit bone formation.

Vitamin D and breast cancer risk.

In addition to its important role in the maintenance of the skeleton, there is mounting evidence that vitamin D has effects on other body systems, and that adequate supplies of vitamin D are likely to be required for optimal health. Vitamin D is obtained both from dietary sources and from cutaneous synthesis with exposure to sunlight. Some epidemiological studies have indicated that vitamin D deficiency and decreased exposure to solar UVB radiation increase the risk of some cancers, including breast cancer. The active metabolite of vitamin D, 1,25-dihydroxy-vitamin D(3), is synthesized primarily in the kidney, and has been shown in laboratory studies to have potent anti-proliferative effects on breast cancer cells. Normal and neoplastic breast tissues contain the vitamin D receptor, and gene ablation studies have implicated the receptor in normal breast development. Several polymorphisms have been identified in the vitamin D receptor gene, and these have been associated with risk of breast cancer in some studies. Local synthesis of 1,25-dihydroxyvitamin D(3) in breast tissue may contribute to maintenance of normal cell function, which could be impaired in vitamin D deficiency.

Doxycycline induces caspase-dependent apoptosis in human pancreatic cancer cells.

Doxycycline (DC) belongs to the tetracycline family of antibiotics and has been used clinically for over 5 decades. Despite advances in understanding the molecular pathogenesis of pancreatic cancer, no chemotherapy course has shown significant effectiveness. Hence new treatments are needed. In this study we report the pro-apoptotic effects of DC in 2 pancreatic adenocarcinoma cell lines, T3M4 and GER. Cell proliferation was measured using the SRB protein dye. Induction of apoptosis was detected using ELISA. Caspase activation was detected using either immunoblotting or a colorimetric assay based on cleavage of caspase-associated substrates. Expression of proteins and post-translational modifications were determined using immunoblotting. Treatment of pancreatic cancer cells with DC reduces their proliferation. This reduction is, at least partly, due to increased caspase-dependent apoptosis involving activation of caspase3, caspase7, caspase8, caspase9, caspase10 and increased levels of FADD. Inhibition of caspase8 or caspase10 but not caspase9 significantly decreases DC-induced apoptosis in both cell lines. Furthermore treatment of pancreatic cancer cells with DC increases protein levels of Bax and phosphorylation of members of the p38MAPK pathway such as p38MAPK, MKK3/6 and MAPKAPK2. These results provide an insight into mechanisms behind the pro-apoptotic effects of DC in pancreatic cancer cells.

Epigenetic corruption of VDR signalling in malignancy.

BACKGROUND: The ligand-mediated switch from binding co-repressor to co-activator complexes is central to the transcriptional actions of the vitamin D receptor (VDR) and other nuclear receptors. The capacity of deregulated co-repressors to attenuate the responsiveness of VDR signalling in cancer models was examined. MATERIALS AND METHODS: Proliferation and gene regulation studies were undertaken in non-malignant and malignant cell line and primary models. RESULTS: Both primary tissue models and cancer cell lines displayed a spectrum of suppressed responsiveness towards 1alpha, 25 hydroxy vitamin D3 (1alpha25(OH)2D3) which correlated with elevated co-repressor content: specifically, elevated silencing mediator of retinoid and thyroid hormone receptors/nuclear co-repressor 2 (NCoR2/SMRT) in prostate cancer cell lines and primary tumour cultures, and elevated nuclear receptor co-repressor 1 (NCoR1) in breast cancer cell lines. Interestingly, whilst the cancer cell lines frequently also displayed reduced VDR content, the primary tumour material retained and/or elevated VDR mRNA, correlated with co-repressor content. Functional approaches towards NCoR2/SMRT (siRNA) in prostate cancer cells or NCoR1 (overexpression) in non-malignant breast epithelial cells confirmed a role in suppressing VDR transcriptional and cellular actions. Targeted co-treatments of 1alpha25(OH)2D3 plus HDAC inhibitors (TSA, NaB) resulted in re-expression of antiproliferative target genes (e.g., GADD45alpha, p21(waf1/cip1)) and synergistic inhibition of proliferation. CONCLUSION: These data suggest that VDR actions in solid tumours are retained, but were skewed by epigenetic mechanisms to suppress selectively antiproliferative target gene promoter responses. This molecular lesion provides a novel chemotherapy target for acceptable doses of 1alpha25(OH)2D3 plus HDAC inhibitors.

Vitamin D status and breast cancer risk.

BACKGROUND: Local synthesis of 1alpha,25(OH)D3 in breast tissue may contribute to maintenance of normal cell function and could be impaired with low circulating levels of the precursor 25hydroxyvitamin D. The aims of this study were to: i) assess the association between breast cancer risk and plasma 25OHD3 concentration and ii) define the significance of expression of the 25OHD activating enzyme CYP27b1 in non-malignant and malignant models of breast epithelial cells. MATERIALS AND METHODS: Breast cancer patients and control women were recruited and their 25OHD levels measured by enzyme-linked immunosorbent assay (ELISA). MRNA expression of CYP271b and the 1,25(OH)2D3 inactivating enzyme CYP24 were measured in breast cancer cell lines by RT-PCR and correlated with immunoblotting approaches to the translated proteins. RESULTS: For women with 25OHD < 50 nM the odds ratio for breast cancer compared with women with 25OHD > 50 nM was 3.54 (CI 1.89-6.61, p < 0.001). CYP271b and CYP24 were detected in non-malignant and malignant cell models. Protein levels of 24OHase but not 1alphaOHase were decreased at confluence in the cell lines. CONCLUSION: Impaired local generation of 1,25OHD3 may contribute to the development of breast cancer.

Altered nuclear receptor corepressor expression attenuates vitamin D receptor signaling in breast cancer cells.

PURPOSE: We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1alpha,25-dihydroxyvitamin D(3). EXPERIMENTAL DESIGN: Profiling, transcriptional, and proliferation assays were undertaken in 1alpha,25(OH)(2)D(3)-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21). RESULTS: Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G(1) of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor alpha-negative (n = 7) tumors. 1alpha,25(OH)(2)D(3) resistance in cancer cell lines was targeted by cotreatments with either 1alpha,25(OH)(2)D(3) or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D(3) compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells. CONCLUSIONS: Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1alpha,25(OH)(2)D(3) resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D(3) compounds and low doses of TSA.

Investigation of the mechanisms by which EB1089 abrogates apoptosis induced by 9-cis retinoic acid in pancreatic cancer cells.

OBJECTIVES: Previous research has shown that the retinoid 9-cis retinoic acid (RA) promotes apoptosis in pancreatic cancer cells. The vitamin D analog EB1089 does not. Furthermore, cotreatment of cells with 9-cis RA and EB1089 abrogates apoptosis. To explain this, we studied the regulation of proteins involved in apoptotic signaling pathways in pancreatic cancer cells. METHODS: The pancreatic adenocarcinoma cell line T3M4 was used. Cell proliferation was measured using the SRB protein dye assay. Induction of apoptosis was evaluated using an ELISA assay. Caspase activation was detected using a colorimetric assay based on cleavage of a caspase-associated substrate. Regulation of protein levels and posttranslational events were detected using immunoblotting. RESULTS: We confirm that EB1089 diminishes apoptosis induced by 9-cis RA in T3M4 cells. We extend the study to show that EB1089 abrogates increases, induced by 9-cis RA, in caspase activation, p27Kip1 protein levels, Bim and Bax protein levels and in Bax/Bcl2 ratio. In addition, the CDKI p21Waf1 and CAII, a differentiation marker for pancreatic cancer cells are also differentially regulated. CONCLUSIONS: These results suggest that the inhibitory effects of EB1089 on 9-cis RA-induced apoptosis lie upstream of caspase activation and could be associated with reduction of p27Kip1 protein levels.

Biological actions of extra-renal 25-hydroxyvitamin D-1alpha-hydroxylase and implications for chemoprevention and treatment.

The Vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase) is now known to be expressed in a much wider range of tissues that previously thought, suggesting a role for 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), which is more in keeping with a cytokine than a hormone. In this capacity, the function of 1alpha-hydroxylase in tumors is far from clear. Studies from several groups including ours have shown altered expression of 1alpha-hydroxylase in different types of neoplasm including breast, prostate and colon cancers. However, functional analysis of Vitamin D metabolism in cancer is complicated by the heterogenous composition of tumors. Immunohistochemical analysis of breast tumors has shown that 1alpha-hydroxylase is expressed by both epithelial cells and by tumor-infiltrating macrophages, suggesting an immunomodulatory component to 1,25(OH)(2)D(3) production in some types of cancer. The demonstration of 1alpha-hydroxylase activity in tumors and their equivalent normal tissues has implications for both the treatment and prevention of cancers. For example, in tumors chemotherapy options may include the use of non-1alpha-hydroxylated Vitamin D analogs to increase local concentrations of active metabolites without systemic side-effects. The role of 1alpha-hydroxylase in protection against cancer is likely to be more complicated and may involve anti-tumor immune responses.

Autocrine metabolism of vitamin D in normal and malignant breast tissue.

PURPOSE: Vitamin D seems to exert a protective effect against common cancers, although this does not correlate with circulating levels of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], indicating a more localized activation of vitamin D. The aim of this study was to investigate the significance of this in breast cancer. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR analysis of mRNA expression was carried out for the vitamin D-activating enzyme 1alpha-hydroxylase, the catabolic enzyme 24-hydroxylase, and the vitamin D receptor in 41 tumors and paired nonneoplastic tissue as well as breast cancer cell lines. Immunohistochemistry was used to assess 1alpha-hydroxylase protein expression, and enzyme assays were used to quantify vitamin D metabolism. RESULTS: Expression of mRNA for 1alpha-hydroxylase (27-fold; P < 5 x 10(-11)), vitamin D receptor (7-fold; P < 1.5 x 10(-8)), and 24-hydroxylase (4-fold; P < 0.02) was higher in breast tumors. 1alpha-Hydroxylase enzyme activity was also higher in tumors (44.3 +/- 11.4 versus 12.4 +/- 4.8 fmol/h/mg protein in nonneoplastic tissue; P < 0.05). However, production of inactive 1,24,25-trihydroxyvitamin D3 was also significantly higher in tumors (84.8 +/- 11.7 versus 33.6 +/- 8.5 fmol/h/mg protein; P < 0.01). Antisense inhibition of 24-hydroxylase in vitro increased antiproliferative responses to 1,25(OH)2D3. CONCLUSION: These data indicate that the vitamin D-activating enzyme 1alpha-hydroxylase is up-regulated in breast tumors. However, dysregulated expression of 24-hydroxylase seems to abrogate the effects of local 1,25(OH)2D3 production in tumors by catalyzing catabolism to less active vitamin D metabolites. The enzymes involved in autocrine metabolism of vitamin D in breast tissue may therefore provide important targets for both the prevention and treatment of breast cancer.

Plasma 25-hydroxy vitamin D concentrations, vitamin D receptor genotype and breast cancer risk in a UK Caucasian population.

Low levels of 25-hydroxy vitamin D (25(OH)D) and polymorphisms in the vitamin D receptor gene (VDR) have been found separately to increase risk of breast cancer. The aim of this study was to determine whether low 25(OH)D levels, alone and in combination with BsmI VDR genotype, increased breast cancer risk in a United Kingdom (UK) Caucasian population. Breast cancer patients (n=179) and control women (n=179) were recruited and 25(OH)D levels measured by enzyme-linked immunosorbent assay (ELISA). VDR genotype was determined by polymerase chain reaction (PCR) and restriction enzyme digest. Analysis showed that subjects with 25(OH)D levels <50 nM and the bb BsmI VDR genotype are 6.82 times more likely to have breast cancer than subjects with levels of 25(OH)D>50 nM and either the BB or Bb genotype (95% confidence interval (CI) 2.31-14.7, P<0.001). This study indicates that low levels of circulating 25(OH)D, both alone and in combination with BsmI VDR genotype, may increase risk of breast cancer in a UK Caucasian population.

Induction of apoptosis in breast cancer cells by apomine is mediated by caspase and p38 mitogen activated protein kinase activation.

The 1,1-bisphosphonate ester family member apomine (SR-45023A) is known to have anti-tumour activity in various cancer cell types. The aims of this study were to determine the effect of apomine on the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231, to ascertain whether any growth inhibitory effects found were due to induction of apoptosis, and to investigate the mechanism of action of apomine. Apomine caused significant growth inhibition of both cell lines after 72h of treatment. Apomine-induced growth inhibition was associated with caspase and p38 MAPK activation and DNA fragmentation. Apomine had no effect on Ras localisation, nor did addition of mevalonate to treatment media prevent apomine-induced apoptosis. We conclude that apomine induces apoptosis in breast cancer cells, an effect that is independent of oestrogen receptor status and is not via inhibition of the mevalonate pathway. Our study suggests apomine is a potential anti-neoplastic drug in breast cancer treatment.

Vitamin D receptor gene polymorphisms and breast cancer risk.

PURPOSE: The steroid hormone 1,25-dihydroxyvitamin D3 is thought to protect against breast cancer. The actions of 1,25-dihydroxyvitamin D3 are mediated via the vitamin D receptor (VDR), and a number of polymorphisms in the VDR gene have been identified. These result in distinct genotypes, some of which may alter susceptibility to breast cancer. We have investigated whether specific VDR gene polymorphisms are associated with breast cancer risk in a United Kingdom Caucasian population. EXPERIMENTAL DESIGN: In a retrospective case-control study, female breast cancer patients (n = 398) and control women (n = 427) were recruited, and three VDR polymorphisms were determined. RESULTS: The 3' VDR polymorphisms BsmI and variable-length poly(adenylate) sequence were both significantly associated with breast cancer risk; odds ratios (adjusted for age menopausal status and hormone replacement therapy usage) for bb genotype versus BB genotype = 1.92 (95% confidence interval, 1.20-3.10; P < 0.01) and for LL versus SS = 1.94 (95% confidence interval, 1.20-3.14; P < 0.01). A 5' VDR gene variant, FokI, was not associated with breast cancer risk when analyzed in isolation (P > 0.05). However, FokI did modulate the increased risk associated with the bb/LL genotype such that possession of one or more F alleles together with the bb/LL genotype augmented breast cancer risk. Furthermore, the highest proportion of bb and FFLL/FfLL genotypes occurred in women with metastatic breast cancer. CONCLUSIONS: VDR polymorphisms are associated with breast cancer risk and may be associated with disease progression. Additional investigations into how different genotypes may affect the functional mechanisms of the VDR will provide a better strategy for identifying women at risk of breast cancer and for developing improved treatments.

Zoledronic acid induces apoptosis and inhibits adhesion to mineralized matrix in prostate cancer cells via inhibition of protein prenylation.

OBJECTIVE: To investigate effects of zoledronic acid on apoptosis and adhesion to mineralized matrix in prostate cancer cells, to quantify these actions, and to elucidate some of the underlying molecular mechanisms, in terms of dependence on caspase activation and involvement of protein prenylation. MATERIALS AND METHODS: DU145 and PC-3 prostate cancer cell lines were used; cells were treated with zoledronic acid, with or without several other reagents, to investigate its mechanism of action. Apoptosis was detected using a cell-death detection enzyme-linked immunosorbent assay. Adhesion was measured by seeding cells onto mineralized dentine inserts for 24 h, and counting cells after washing. RESULTS: Apoptosis depended on time and dose; there was significant apoptosis with higher concentrations of zoledronic acid (100 micromol/L) after 24 h of exposure, and in DU145 cells with concentrations as low as 1 micromol/L after 72 h of exposure. The apoptotic effect was diminished by co-treating with a broad-spectrum caspase inhibitor, Z-VAD-FMK. Zoledronic acid at 1 micromol/L also significantly inhibited cell adhesion to the mineralized matrix. The lipid isoprenoid analogue geranylgeraniol reduced the apoptotic and anti-adhesive effects of zoledronic acid to a greater degree than farnesol. There was also apoptosis and inhibition of adhesion with direct inhibitors of prenylation, e.g. manumycin A and GGTI-298. C3 exoenzyme, an inhibitor of RhoA, inhibited adhesion but did not cause apoptosis. CONCLUSION: Zoledronic acid induces apoptosis in prostate cancer cells via a caspase-dependent mechanism, and at concentrations as low as 1 micromol/L it also inhibits adhesion of cells to mineralized matrix. These effects appear to be exerted via inhibiting G-protein prenylation and in particular geranylgeranylation.

Approaches to evaluating the association of vitamin D receptor gene polymorphisms with breast cancer risk.

The steroid hormone 1,25 dihydroxyvitamin D3 is thought to protect against breast cancer. Its actions are mediated via the vitamin D receptor (VDR) and a number of polymorphisms in the VDR gene have been identified, some of which may alter susceptibility to breast cancer. This study has investigated whether specific VDR gene polymorphisms are associated with breast cancer risk in a UK Caucasian population. Female breast cancer patients (n = 313) and control women with a negative screening mammogram (n = 410) were recruited and their VDR polymorphisms were determined. The 3' VDR polymorphism BsmI was significantly associated with breast cancer risk; odds ratio bb vs. BB genotype = 1.79 (95% CI, 1.12-2.86; P = 0.0221). In addition, over 70% of seven commonly used breast cancer cell lines were found to have the at-risk genotype bb. The 5' FokI gene variant was not associated with breast cancer risk. Further investigations into how these different genotypes may affect the functional mechanisms of the VDR will provide a better strategy for identifying women at risk of breast cancer and for developing improved treatments.

Mechanisms implicated in the growth regulatory effects of vitamin D compounds in breast cancer cells.

The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)D3), has been recognized for over 2 decades as a modulator of cell proliferation and differentiation in many cell types, including breast cancer. However, any potential anti-tumour properties displayed by 1,25(OH)D3 are limited by the tendency to cause hypercalcaemia when administered at high doses. Because of this, synthetic vitamin D analogues have been developed that retain the anti-tumour effects seen with 1,25(OH)D3 but which have reduced calcaemic activity. However, it is still unclear as to how 1,25(OH)D3 and its synthetic analogues act within breast cancer cells to elicit the effects on cellular proliferation and differentiation. In this chapter we review the advances that have been made in trying to answer this question. It has been found so far that 1,25(OH)D3 has an effect on the expression of certain cell cycle regulators and in this way can bring about G1 arrest. Evidence has also emerged that vitamin D compounds can also affect the growth-promoting pathways initiated by two important factors involved in breast cancer cell promotion; namely the insulin-like growth factor I (IGF-I) and oestrogen-receptor (ER) pathways. Vitamin D compounds have also been implicated in promotion of apoptosis in breast cancer cells and evidence suggests that 1,25(OH)D3 and its synthetic analogues may potentiate responsiveness of breast cancer cells to conventional cytotoxic agents. Although much remains to be learned about the associated underlying mechanisms, ongoing research suggests that vitamin D analogues are a new class of compounds with potential in breast cancer treatment and prevention.

Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway, impairing Ras membrane localization in prostate cancer cells.

PURPOSE: Metastasis to bone is an important cause of morbidity in advanced prostate cancer. Despite the typically sclerotic nature of prostatic bone metastases osteolysis has a significant role in the pathogenesis of this disease. The nitrogen containing bisphosphonates (N-BPs), such as pamidronate and zoledronic acid, have greatly enhanced potency for inhibiting bone resorption and inducing apoptosis in osteoclasts. We investigated the effects of N-BPs on prostate cancer cells. MATERIALS AND METHODS: Cell viability was determined with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay. Cell cycle analysis, DNA fragmentation and caspase 3 activity were assessed using flow cytometry. Ras, Bcl-2 and Bax were quantified by Western blotting. RESULTS: Pamidronate and zoledronic acid decreased cell viability in the 3 human cell lines DU145, PC3 and LNCaP. These effects were associated with changes in cell cycle distribution, induction of DNA fragmentation and a decrease in the Bcl-2-to-Bax ratio, which are features of apoptotic cell death. Pre-incubation with caspase inhibitors attenuated the effects of zoledronic acid and caspase 3 activity was demonstrated in treated DU145 cells. Zoledronic acid induced loss of cell viability in DU145 cells was prevented by co-treatment with farnesol, suggesting that N-BPs cause inhibition of the mevalonate pathway and Ras prenylation. A decrease in active, membrane bound Ras in zoledronic acid treated DU145 cells was shown by Western blot analysis. CONCLUSIONS: N-BPs induce apoptosis in prostate cancer via a caspase dependent mechanism. They have effects on protein prenylation via inhibition of the mevalonate pathway and impair membrane localization of Ras in prostate cancer cells.

Direct effects of bisphosphonates on breast cancer cells.

In addition to inhibiting bone resorption, bisphosphonates have also been shown to exhibit antitumour effects. In vitro, bisphosphonates inhibit proliferation and induce apoptosis in cultured human breast cancer cells. In addition, bisphosphonate treatment interferes with breast cancer cell adhesion to bone matrix, and inhibits cell migration and invasion. The combination of bisphosphonates with other anticancer drugs such as the taxoids markedly enhances these effects. These newly recognized direct actions of bisphosphonates on breast cancer cells indicate that these agents may have a greater role to play in treatment of patients suffering from cancers with a propensity to metastasize to bone.