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Keywords:

  • vitamin D;
  • calcium-sensing receptor;
  • human colon carcinoma cells;
  • drug sensitivity

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Vitamin D (VD) protects against colon carcinogenesis by mechanisms not fully understood. We had earlier reported on the similarity in the biologic action of VD and that of the calcium-sensing receptor (CaSR) in human colon carcinoma cells. At the molecular level, the CaSR gene contains 2 VD response elements and VD stimulates the expression of CaSR. In this study, we investigated on the relationship between VD action and CaSR function. We determined and compared the action of VD in human colon carcinoma cells (CBS, Moser, Caco-2 and HCT116) and their CaSR knocked-down counterparts. VD inhibited cellular proliferation, cellular invasion, and anchorage-independent growth and stimulated the expression of p21/Waf1 but not in CaSR knocked-down cells. These results demonstrate, for the first time, that the known tumor-suppressive function of VD requires functional CaSR and knocking down CaSR expression abrogated this function of VD. We recently reported that activation of CaSR in human colon carcinoma cells downregulated the expression of thymidylate synthase (TS) and survivin and promoted a significant increase in sensitivity to cytotoxic drugs. We now demonstrate, for the first time, that VD suppressed the expression of TS and survivin, TS and survivin gene transcriptional activities and promoted a cytotoxic response to 5-FU in a CaSR-dependent manner. Ectopic expression of wild-type CaSR in colon carcinoma cells also inhibited the expression of TS and survivin and enhanced cellular sensitivity to 5-FU. VD, however, could no longer enhance cellular sensitivity to 5-FU in cells overexpressing CaSR.

Vitamin D (VD) is a chemoprotective agent against colorectal carcinogenesis though its mechanisms of action are not fully understood.1–3 An inverse relationship exists between sunlight exposure and colorectal cancer mortality.4 It has been hypothesized that increased sunlight exposure protects against colorectal cancer through the increased synthesis of VD and its active metabolite, calcitriol, in the skin.4 Case control studies have also demonstrated a linkage between VD deficiency and colorectal cancer.3–5In vitro, VD inhibits cellular proliferation and induces differentiation in human colon carcinoma cells.6–8 At the molecular level, VD stimulates the expression of the tumor suppressor and intercellular adhesion molecule E-cadherin, induces the expression of the cyclin-dependent kinase inhibitor, p21/Waf1, and suppresses β-catenin/TCF-associated Wnt signal pathways.6, 8 In this regard, the molecular action of VD closely resembles that of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor that controls proliferation and differentiation in the human colonic epithelium and colon carcinoma cells.6, 9–13

Thymidylate synthase (TS) is a key enzyme involved in the de novo synthesis of DNA and is the molecular target of the antimetabolite fluorouracil (5-FU).14–16 5-FU is the main stay and a drug of choice used in combination with others in a therapeutic regimen for treating colon cancer.17 5-FU blocks TS reaction, disrupts DNA synthesis and induces cell death.14, 15 Thus, increased TS expression circumvents the efficacy of 5-FU and many cancer types express a high level of TS.15, 16 Increased TS expression is an underlying mechanism of resistance to 5-FU in colon cancer.15, 16 Survivin is a key antiapoptotic protein and its expression in cancer cells blocks the ease with which cell death occurs in response to treatment with cytotoxic drugs.18, 19 Expression of survivin is an overall underlying mechanism of anticancer drug resistance.18, 19 We recently reported that activation of CaSR, in human colon carcinoma cells, downregulated the expression of TS, the expression of survivin and promoted a significant increase in cellular sensitivity to 5-FU.10

The human CaSR gene has 2 promoters (P1 and P2) and each promoter has a transcriptional start site containing a VD response element.20, 21 VD upregulates CaSR mRNA expression in parathyroid, thyroid and kidney tissues in the rat.20, 21 VD induces P1 and P2 CaSR promoter transcriptional activity and CaSR protein expression in human colon carcinoma cells.6 Because of the linkage of VD to CaSR at the molecular level and the similarity of the reported action of VD and CaSR in human colon carcinoma cells,6 we hypothesize that some of the biologic action of VD is linked to the action of CaSR. In this study, we first determined if VD mediated its known growth-inhibitory and tumor-suppressive function in human colon carcinoma cells through the CaSR. We then determined if VD could modulate the expression of TS and survivin, cellular response to 5-FU and the role of CaSR, if any, in such modulation.

Using an experimental approach utilizing shRNA targeting the CaSR, we demonstrate, for the first time, that VD mediated its known growth-inhibitory and tumor-suppressive function in a CaSR-dependent manner. We also demonstrate, for the first time, that VD downmodulated the expression of TS and survivin and promoted a cytotoxic response to 5-FU in a CaSR-dependent manner. Thus, knocking down CaSR expression abrogated the biologic action of VD. In addition, ectopic expression of wild-type CaSR in human colon carcinoma cells suppressed the expression of TS and survivin and enhanced cellular sensitivity to 5-FU. However, VD could no longer further enhance cellular sensitivity to 5-FU in cells overexpressing CaSR.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture, treatment with VD and 5-FU, cell viability assay

Human colon carcinoma cell lines CBS, Moser, Caco-2 and HCT116 and their corresponding shRNA-CaSR (and scrambled control) transfected counterparts were maintained in SMEM medium (Sigma, St Louis, MO) supplemented with sodium bicarbonate, peptone, vitamins, amino acids and 5% fetal bovine serum (FBS) as described previously.6, 10, 11 To determine the effect of VD on the cytotoxic response to 5-FU, the medium of actively growing cells was replenished with medium containing 1 μM calcitriol (active metabolite of VD) and 15 μM 5-FU. The medium of a parallel set of cultures was replenished with medium containing 5-FU without the addition of exogenous calcitriol. The medium of control cultures was replenished only with regular culture medium without 5-FU or calcitriol. Cells were then incubated at 37°C in a CO2 incubator for the time periods as indicated in the figure legends. At the end of an incubation period, cells were detached from the culture wells by trypsinization and suspended in phosphate-buffered saline (PBS). A Vi-Cell™ XR Cell Viability Analyzer (Beckman Coulter, Fullerton, CA) was used to determine the number of viable cells in a suspension. 5-FU was purchased from Sigma (St Louis, MO). Calcitriol was purchased from Cayman Chemical (Ann Arbor, MI).

Cell transfection

shRNA-CaSR expression vector was constructed as previously described.10 Plasmids of shRNA-CaSR or shRNA-scrambled control were transfected into CBS, Moser, Caco-2 and HCT116 cells, and stable shRNA-CaSR or shRNA-scrambled transfectants were established by selection and maintenance of the cells with antibiotics as previously described.10 Transfectants were routinely cultured in SMEM medium containing 50 μg/mL hygromycin B.10 The 4 human colon carcinoma cell lines were also transiently transfected with either a wild-type CaSR expression plasmid (WT-CaSR) or a control empty vector (EV) using Lipofectamine™ 2000 (Invitrogen Life Technologies, Frederick, MD) according to the manufacturer's protocol. WT-CaSR plasmid, a gift from Dr. G. N. Hendy of McGill University (Montreal, Canada), was a construct containing full length CaSR cDNA tagged with c-Myc (amino acids 408–439 of c-Myc) and cloned into pCDNA3.1.22 Cells were washed and adapted in antibiotic-free medium for 24 hr before experimentation.

Cell proliferation assay

The effect of VD on cellular proliferation was determined using the 4 parental cell lines and their corresponding shRNA-CaSR (and scrambled control) transfectants. Proliferation assays were performed as previously described.12 In brief, cells were plated in 24-well cell culture plates at 1 × 104 cells per well in SMEM containing 2.5% FBS. After allowing attachment overnight, the cells were washed twice with serum-free SMEM and incubated in SMEM with 2.5% FBS in the absence or presence of 1.0 μM calcitriol. The culture medium of each culture plate was changed with fresh medium (with and without calcitriol) every 24 hr. Cellular proliferation was measured by detaching the cells with trypsin/EDTA and counting the number of cells automatically using a Vi-Cell™ XR Cell Viability Analyzer (Beckman Coulter, Fullerton, CA). Cell counts were performed at 24, 48 and 72 hr after the cells were plated. The results presented represent the mean and standard error of triplicate determinations.

Western blot analysis

Western blottings were performed essentially as previously described.6, 11 Cells were lysed in lysis buffer [50 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mmol/L sodium orthovanadate, 10 μL/mL protease inhibitor cocktail and 1 mmol/L PMSF] by incubating for 20 min on ice. Protein concentration was determined by the Bio-Rad assay system (Bio-Rad, Hercules, CA). Total proteins were fractionated by SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was blocked with 5% nonfat-dried milk in 1× TBS buffer containing 0.1% Tween 20 and then incubated with appropriate primary antibodies. Antihuman CaSR antibody was purchased from Millipore (Billerica, MA). Antihuman TS antibody was purchased from Zymed Laboratories (Carlsbad, CA). Antihuman p21/Waf1 antibody was purchased from Cell Signaling (Danvers, MA). Antihuman survivin antibody was purchased from R&D Systems (Minneapolis, MN). Antihuman c-Myc antibody was purchased from Cell Signaling (Danvers, MA). Horseradish peroxidase-conjugated antirabbit, antimouse or antigoat IgG was used as the secondary antibody and the protein bands of interest were detected by using the FUJIFILM LAS-3000 system (Fujifilm Life Science, Stamford, CT). β-actin (Abcam, Cambridge, MA) was used as internal controls to evaluate the uniformity of protein loading.

Quantitative analysis of protein expression was performed using Multi Gauge-Image software installed in the FUJIFILM LAS-3000 system. Densitometric measurements in the increase or decrease in protein expression, by comparison with control lanes, was calculated. The numbers on the blots represent the levels of protein expression by comparison with control lanes with an assigned value of 1.

Luciferase reporter assays

Cell lines and their shRNA-CaSR stable (and scrambled control) transfectants were seeded in 24-well culture plates and grown to 70–80% confluence. Cells were transfected with 0.7 μg of either survivin promoter luciferase reporter plasmid pLuc-2840 or TS promoter luciferase reporter plasmid 3RG and 0.1 μg of Renilla luciferase reporter control plasmid per well using Lipofectamine™ 2000 (Invitrogen Life Technologies, Frederick, MD), as previously described.6, 11, 22 Calcitriol (1.0 μM) was added 24 hr after transfections, and luciferase activities were measured at 24 hr after calcitriol treatment. Transfection efficiencies were determined by Renilla luciferase activities. Survivin promoter luciferase reporter plasmid pLuc-2840 was a gift from Dr. F. Li,23 and TS promoter luciferase reporter plasmid 3RG was a gift from Dr. R. D. Ladner.24

Invasion assay

Cell invasion assay were performed using 24-well BD Biocoated Matrigel invasion chambers with 8-μm polycarbonated filters (BD Biosciences, Bedford, MA), as described previously.25, 26 In brief, the 4 cell lines and their shRNA-CaSR (and scrambled control) transfectants were seeded into Matrigel invasion chamber with 105 cells per well. The cells were cultured in 5% FBS/SMEM medium in the absence or presence of 1.0 μM calcitriol for 24 hr. Invasive cells that had penetrated through the matrigel and migrated to the underside of the membrane or into the bottom wells were counted under microscopic vision after fixation with 4% formaldehyde in PBS. The results presented represent the mean and standard error of triplicate determinations.

Anchorage-independent clonogenic assay

This assay was performed as previously described.26, 27 In brief, 104 cells of the 4 cell lines and their shRNA-CaSR (and scrambled control) transfectants were resuspended in 0.6 mL of 0.3% agarose gel (Invitrogen, Carlsbad, CA) in 10% FBS/SMEM medium alone (controls) or in the same medium containing 15 μM 5-FU or in medium containing 5-FU and 1.0 μM calcitriol. The cell–agar mixture was immediately seeded into 24-well plates precoated with their corresponding 0.6% agar in control 10% FBS/SMEM medium or medium containing 5-FU or medium containing both 5-FU and calcitriol. The culture plates were then incubated in a 37°C, 5% CO2 incubator for 2–4 weeks, and the number of colonies formed was scored under microscopic vision. The results presented represent the mean and standard error of triplicate determinations.

Statistical analysis

Mean data of experiments are given as mean ± SE. Statistical significance was tested with unpaired Student's t test. p ≤ 0.01 indicates a significant difference by comparison with control values.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

VD inhibits cellular proliferation and invasion and induces p21/Waf1 expression in a CaSR-dependent manner

A shRNA-CaSR plasmid was constructed to probe the relationship between VD action and the function of CaSR. As expected, all the colon carcinoma cell lines constitutively expressed CaSR (Fig. 1a, lane 1). The expression of CaSR was significantly reduced in cells stably transfected with the shRNA-CaSR plasmid (Fig. 1a, lane 3), but not in cells transfected with the control plasmid (Fig. 1a, lane 2). Using the parental cells, their shRNA-CaSR-transfected and control-transfected counterparts as a model, we first determined the role of CaSR in mediating the known action of VD with regard to the inhibition of cellular proliferation, invasion and the induction of the expression of p21/Waf1. As expected, inclusion of VD (1.0 μM calcitriol) in the culture medium effectively inhibited cellular proliferation (Fig. 1b). However, VD was not effective in inhibiting the proliferation of shRNA-CaSR-transfected cells with downregulated CaSR expression (Fig. 1b). Likewise, VD inhibited cellular invasion in the parental but not in shRNA-CaSR-transfected cells with downregulated CaSR expression (Fig. 1c). VD is known to induce the expression of p21/Waf1, a key molecule in blocking cellular proliferation, and is regarded as a differentiation marker in colon cancer.28 As expected, VD induced the expression of p21/Waf1 in the parental cells (Fig. 1d). The ability of VD to induce the expression of this molecule, however, was significantly reduced in CaSR knocked down cells (Fig. 1d). These results show that these known biologic actions of VD are linked to CaSR function.

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Figure 1. Linkage of VD action to CaSR. (a) Transfection with shRNA-CaSR knocked down the expression of CaSR. Western blots of CaSR expression in shRNA-CaSR-transfected and control (shRNA-scrambled)-tranfected CBS, Moser, Caco-2 and HCT116 cells. The expression of β-actin was used as internal control for equal protein loading. Lane 1, parental cells. Lane 2, shRNA-scrambled control-transfected cells. Lane 3, shRNA-CaSR-transfected cells. Numbers on blots represent the levels of CaSR expression by comparison to parental cells with an assigned value of 1. (b) Knocking down CaSR expression circumvented the growth-inhibitory effect of VD. The growth rates of parental, shRNA-scrambled-transfected and shRNA-CaSR-transfected cells in control medium or in medium containing calcitriol (1.0 μM) were determined as described in Material and Methods. Results are expressed as mean and standard error of the mean of triplicate experiments. (c) Knocking down CaSR expression blocked the inhibitory effect of VD on cellular invasion. These assays were performed as described in Material and Methods using commercially available invasion chambers. Hollow bars, cells in control medium; solid bars, cells in medium supplemented with 1.0 μM calcitriol; P, parental cells; S, shRNA-scrambled-transfected control cells; C, shRNA-CaSR-transfected cells. Asterisk (*) indicates p < 0.01 compared to control cells (hollow bars). (d) Knocking down CaSR expression blocked the induction of p21/Waf1 by VD. Western blot analysis of cells cultured in control medium or in medium containing calcitriol (1.0 μM). Lane 1, parental cells; lane 2, parental cells exposed to calcitriol for 24 hr; lane 3, shRNA-scrambled transfectants; lane 4, shRNA-scrambled transfectants exposed to calcitriol for 24 hr; lane 5, shRNA-CaSR transfectants; lane 6, shRNA-CaSR transfectants exposed to 1.0 μM calcitriol for 24 hr. β-actin was used as an internal control for equal protein loading. Numbers on top of each band indicate the levels of protein expression in comparison to controls (lane 1).

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VD promotes cellular sensitivity to 5-FU in a CaSR-dependent manner

We recently reported that activation of CaSR in human colon carcinoma cells promotes sensitivity to 5-FU.10 Because the promoters of the CaSR gene contain VD response elements6 and VD can stimulate CaSR transcriptional activities and protein expression in these cells, we determined if VD could promote sensitivity to 5-FU. Clonogenic assays performed in soft agar and cytotoxicity assays in monolayer cultures were used to measure the cellular response to 5-FU. Figure 2a shows that VD promoted the suppressive effect of 5-FU on colony formation. In the presence of VD, colony formation was significantly further inhibited by 5-FU in comparison to the inhibitory effect exerted by 5-FU alone (Fig. 2a). VD, however, could not enhance the inhibitory effect of 5-FU in CaSR knocked down cells (Fig. 2a). Likewise, VD promoted cytotoxic responses to 5-FU in these colon carcinoma cells but not in their CaSR knocked down counterparts (Fig. 2b). Thus, VD promoted cellular sensitivity to 5-FU in a CaSR-dependent manner.

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Figure 2. VD enhances cellular responses to 5-FU via CaSR. (a) VD enhances the suppressive effect of 5-FU on anchorage independent growth in parental and shRNA-scrambled-transfected control cells but not in CaSR knocked down cells. Clonogenic assays were performed as described in Material and Methods, in control medium or in medium supplemented with 1.0 μM calcitriol. Hollow bars, cells in agar gel containing 5-FU (15 μM); solid bars, cells in agar gel containing 5-FU (15 μM) and calcitriol (1.0 μM); P, parental cells; S, shRNA-scrambled-transfected control cells; C, shRNA-CaSR-transfected cells. Asterisk (*) indicates p < 0.01 compared with parental cells cultured in medium containing 5-FU but no calcitriol (hollow bars). Error bars represent the mean and standard error of the mean of triplicate experiments. (b) VD enhances the cytotoxic response to 5-FU, in monolayer culture, in a CaSR-dependent manner. Cells in 24-well plate were exposed to 5-FU (15 μM) for 24 hr in culture medium without or with calcitriol (1.0 μM). Viable cells were counted as described in Material and Methods. Results are expressed as percent cells killed by comparison with control cells cultured in the absence of 5-FU and calcitriol. Triplicate determinations were performed in each experiment. The results represent the mean and standard error of the mean of 3 independent experiments. Hollow bars, cells in medium containing 5-FU (15 μM); solid bars, cells in medium containing 5-FU (15 μM) and calcitriol (1.0 μM); P, parental cells; S, shRNA-scrambled-transfected control cells; C, shRNA-CaSR-transfected cells.

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VD downmodulates the expression of TS and survivin in a CaSR-dependent manner

Because activation of CaSR promotes cytototoxic responses to 5-FU with a concurrent downmodulation of the expression of TS and survivin and if VD acts through the CaSR, we expect VD would also downmodulate the expression of TS and survivin in the parental cells but not in their CaSR knocked down counterparts. Indeed, treatment of the parental cells with VD downmodulated the expression of TS and survivin but not in their corresponding CaSR knocked down counterparts (Fig. 3). Similar results were observed in TS and survivin gene transcriptional activities. VD significantly suppressed TS (3RG pLuc-2840) and survivin (pLuc-2840) transcriptional activities in the parental cells but not in their corresponding CaSR knocked down counterparts (Fig. 4). Thus, VD modulated the expression of TS and survivin and their gene transcriptional activities in a CaSR-dependent manner.

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Figure 3. Disruption of CaSR expression blocks the inhibitory effect of VD on the expression of TS and survivin. Western blot analysis of TS and survivin expression was performed as described in Material and Methods. Lane 1, parental control cells; lane 2, parental cells exposed to 1.0 μM calcitriol for 24 hr; lane 3, shRNA-scrambled transfectants; lane 4, shRNA-scrambled transfectants exposed to 1.0 μM calcitriol for 24 hr; lane 5, shRNA-CaSR transfectants; lane 6, shRNA-CaSR transfectants exposed to 1.0 μM calcitriol for 24 hr. β-actin was used as an internal control for equal protein loading. Numbers on top of each band indicate the levels of protein expression in comparison to controls (lane 1).

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Figure 4. VD inhibits TS and survivin gene transcription in a CaSR-dependent manner. Luciferase reporter assays for TS and survivin gene transcriptional activities were performed as described in Material and Methods. Exposure of colon carcinoma cells to calcitriol for 24 hr downregulated TS promoter reporter luciferase activities in (a) and survivin promoter reporter luciferase activities in (b) but not in their shRNA-CaSR-transfected counterparts. Hollow bars, cells cultured in control medium; solid bars, cells cultured in medium supplemented with 1.0 μM calcitriol; P, parental cells; S, shRNA-scrambled-transfected control cells; C, shRNA-CaSR-transfected cells. Asterisk (*) indicates p < 0.01 compared with parental cells cultured in control medium (hollow bars). Error bars represent the mean and standard error of the mean of triplicate experiments.

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Overexpression of CaSR downregulates the expression of TS and survivin and promotes a cytotoxic response to 5-FU

Because the human CaSR gene is inducible by VD,6, 7, 20, 21 we hypothesized that the induction of CaSR was a molecular mechanism underlying VD action. Therefore, we determined if ectopic expression of CaSR would mimic the action of VD in terms of modulation of the markers TS and survivin. We also determined if ectopic expression of CaSR would modulate the cellular response to 5-FU. A c-Myc tagged WT-CaSR plasmid22 was used to ectopically express CaSR in the parental cells, which was detected by anti-c-Myc antibody in Western blot analysis (Fig. 5a). Overexpression of CaSR downregulated both TS and survivin expression in comparison to the EV transfected controls (Fig. 5a). Interestingly, overexpression of CaSR in these colon carcinoma cells also increased their cytotoxic responses to 5-FU (Fig. 5b). However, VD could no longer enhance cellular sensitivity to 5-FU in cells overexpressing CaSR (Fig. 5b).

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Figure 5. Ectopic expression of CaSR suppresses TS and survivin expression and enhances the cytotoxic response to 5-FU. (a) Colon carcinoma cells were transfected with either a c-Myc tagged wild-type CaSR plasmid (WT-CaSR) or a control empty vector (EV) as described in Material and Methods. WT-CaSR expression was determined by Western blottings using anti-c-Myc antibodies. Western blottings were used to determine the level of expression of TS and survivin. β-actin was used as an internal control for equal protein loading. Lane 1, EV-transfected control cells; lane 2, cells transfected with wild-type CaSR plasmid. Numbers on the blots represent the levels of expression of TS and survivin in comparison to control lanes. (b) CaSR expression enhances the cytotoxic response to 5-FU. Cells transfected either with a wild-type CaSR plasmid or a control empty vector as described above were exposed to 5-FU (15 μM) for 24 hr in the absence or presence of calcitriol (1.0 μM). Viable cells were counted as described in Material and Methods. Results are expressed as percent cells killed by comparison to control cultures (no drug) and represent the mean and standard error of the mean of 3 independent experiments. Hollow bars, cells transfected with empty vector; solid bars, cells transfected with WT-CaSR; slashed bars, cells transfected with WT-CaSR and cultured at the presence of calcitriol (1.0 μM).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

VD and Ca2+ are chemopreventive agents in colon cancer.1–3 Both VD and extracellular Ca2+ can inhibit cell proliferation and induce differentiation in human colon carcinoma cells.3, 6, 10–13 Extracellular Ca2+ and CaSR constitute a robust ligand/receptor system in regulating proliferation and differentiation in the colonic epithelium and in colon carcinoma cells in vitro.6, 10–13 At the physiologic level, VD promotes the absorption of Ca2+ in the gut.29 Ca2+ may then activate the CaSR and initiate growth and differentiation control cascades as the colonic epithelial cells migrate and differentiate toward the apex of the colonic crypts. We propose this scenario because without CaSR, extracellular Ca2+ is unable to inhibit proliferation and differentiation in human colon carcinoma cells,9, 10 and in the human colonic epithelium, loss of CaSR expression correlates with lack of differentiation and malignant progression.9, 10 Because VD can stimulate the transcription of CaSR gene and protein expression,6, 21, 22 we determined, in this study, the relationship between the biologic action of VD and CaSR function.

We first investigated on the known biologic action of VD with regard to the inhibition of cellular proliferation and invasion, and the induction of the expression of p21/Waf1. Knocking down the expression of CaSR in these colon carcinoma cells circumvented these functional aspects of VD. These results strongly suggest that VD mediates these actions through the CaSR. We recently reported that activation of CaSR by extracellular Ca2+ in human colon carcinoma cells promotes sensitivity to 5-FU with a concurrent downregulated expression of TS and survivin.10 If VD mediates its action through CaSR, we would expect that VD treatment of colon carcinoma cells would also promote sensitivity to 5-FU and downregulate the expression of TS and survivin and that knocking down CaSR expression would abrogate these actions of VD. Indeed, knocking down CaSR expression circumvented the ability of VD to promote sensitivity to 5-FU and downregulate TS and survivin gene transcription and protein expression. Thus, we have for the first time linked the biologic action of VD to CaSR function and identified new important targets of VD in colon cancer, i.e., TS and survivin. Survivin has also been reported as a VD target in head and neck and breast cancer cells.30, 31 Inability of breast cancer cells to respond to VD is due to failure of VD to downregulate survivin expression.30

Drug-resistant human colon carcinoma cells do not express or express little CaSR.10 These resistant cells, however, express abundant amount of TS and survivin, and without CaSR, they are unresponsive to extracellular Ca2+.10 Because both VD and Ca2+ act through CaSR, we determined if ectopic expression of CaSR itself would downregulate the expression of TS and survivin and promote sensitivity to 5-FU. Indeed, ectopic expression of CaSR in these colon carcinoma cells downregulated the expression of TS and survivin and promoted sensitivity to 5-FU. However, VD was not able to further enhance the sensitivity of these CaSR overexpressing cells to 5-FU. It is likely that these cells have reached the threshold of maximum CaSR expression and VD was ineffective in further stimulating CaSR in these cells. On the other hand, loss of CaSR expression in the colon may allow malignant cells to escape from VD control. There could be a progressive decrease in responsiveness to VD as the expression of CaSR decreases. Alternatively, a minimum threshold in CaSR expression may exist in which the cells no longer respond to VD when the threshold is crossed.

The protective effect of VD against colon carcinogenesis is well known.1–3 Patients with colon cancer and those receiving chemotherapy are several-fold more likely to have severe VD deficiency.1, 32, 33 It has been proposed that patients with colorectal cancer, especially those receiving chemotherapy, should be considered for aggressive VD replacement strategies.1, 32, 33 In view of our findings, whether VD replacement would also improve on the therapeutic outcome awaits investigation in a clinical setting. Development of agents that can induce CaSR expression in colon cancer cells may also be helpful in improving therapeutic outcome.

In summary, we showed that VD mediated its known growth-inhibitory action in human colon carcinoma cells in a CaSR-dependent manner. VD also downmodulated the expression of TS and survivin and promoted a significant increase in cellular sensitivity to 5-FU in a CaSR-dependent manner. Ectopic expression of CaSR itself led to a downregulated expression of TS and survivin and increased sensitivity to 5-FU. However, VD was no longer able to modulate chemosensitivity in cells overexpressing CaSR. How VD and CaSR co-operate to regulate target genes is unknown and will require further investigation at the molecular level.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. Fengzhi Li (Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY) for providing survivin promoter luciferase reporter plasmid pLuc-2840, Dr. Robert D. Ladner (Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA) for providing TS promoter luciferase reporter plasmid 3RG and Dr. Geoffrey N. Hendy (McGill University, Montreal, Canada) for providing wild-type CaSR expression plasmid.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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