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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Pharmacology [P. A. H., W-D. Y., R. A. M., R. M. R., C. S. J.], Medicine [C. S. J., D. L. T.], and Surgery [D. L. T.] and the University of Pittsburgh Cancer Institute [C. S. J., D. L. T.], University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We determined that calcitriol inhibits growth of the murine SCC SCCVII/SF (2 , 7) and the growth and metastatic potential of the Dunning rat prostatic adenocarcinoma, Mat-lylu (1) . We subsequently evaluated the use of calcitriol in combination with cytotoxic agents and found that calcitriol synergistically enhanced the antitumor activity of cisplatin and carboplatin in vitro and in vivo, and that these effects were schedule dependent (13) .
In SCC, mechanistic studies revealed that calcitriol induces G0-G1 arrest (13) , a decrease in Rb phosphorylation, an increase in expression of p27, and a decrease in expression of p21 (14) . Expression of p21 is also reduced in SCC tumors harvested from animals treated with therapeutic doses of calcitriol (14) . These findings suggest that p21 down-modulation may be a component of the mechanism by which calcitriol exerts antiproliferative activity.
Recent studies indicate that a reduction in p21 expression sensitizes tumor cells to both DNA-damaging agents (15 , 16) and microtubule-damaging agents such as paclitaxel (17, 18, 19) . In MCF-7 breast carcinoma cells, paclitaxel induces p21 expression; treatment of these cells with antisense p21 oligonucleotides increases paclitaxel cytotoxicity (17) . Similarly, paclitaxel antitumor activity is increased in vitro and in vivo in HCT116 colon carcinoma cells made deficient in p21 expression (18) . Compared with p21+/+ controls, these cells displayed an increase in paclitaxel-induced apoptosis.
Given the ability of calcitriol to decrease p21 expression in SCC in vitro and in vivo and the reported association between reduced expression of p21 and increased sensitivity to paclitaxel, we hypothesized that calcitriol would enhance the antitumor activity and apoptosis-promoting ability of paclitaxel.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9 days later when tumors were palpable. The human prostatic adenocarcinoma cell line PC-3 was obtained from the American Type Culture Collection (Manassas, VA). For in vitro studies, cells were grown in F-12K medium containing penicillin-streptomycin and 10% FCS and 2 mM L-glutamine at 37°C in a humidified atmosphere containing 5% CO2. In vivo, adenocarcinomas were routinely produced by s.c. inoculation of 2 x 106 log-phase tissue culture cells mixed 1:1 with Matrigel (Becton Dickinson, Bedford, MA) in the right flank of each nude mouse (Taconic Farms, Germantown, NY). Studies were initiated when tumors were palpable. Animals were used in accordance with Institutional Animal Care and Use Committee guidelines.
Chemicals and Reagents.
Calcitriol (HoffmannLaRoche, Nutley, NJ) was reconstituted in 100% ethanol and stored protected from light under a layer of nitrogen gas at -70°C. All handling of calcitriol was performed with indirect lighting. Paclitaxel (Taxol; Bristol-Myers Squibb, Princeton, NJ) was purchased as a 6 mg/ml solution in Cremophor EL and was diluted in tissue culture medium or sterile saline just prior to use. The antibodies used in these studies were monoclonal mouse anti-PARP (Enzyme Systems, Livermore, CA), monoclonal mouse antihuman Bcl-2 (Dako, Carpinteria, CA), and polyclonal rabbit anti-p21 (Santa Cruz Biotechnology, Santa Cruz, CA). Antirabbit and antimouse horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Life Sciences (Arlington Heights, IL) and Promega Corp. (Madison, WI), respectively. Actin was detected using the actin (Ab-1) kit from Oncogene Research Products (Boston, MA).
In Vitro Clonogenic Tumor Cell Survival Assay.
Tumor cells were incubated for 24 h in T25 flasks (Corning Costar Corp., Cambridge, MA) with or without calcitriol. Cells were then either left untreated or were treated with various concentrations of paclitaxel for an additional 24 h. The cells were then harvested and counted, and a fixed number of cells were replated into six-well tissue culture plates (Corning Costar). After a 7-day incubation at 37°C in a humidified atmosphere containing 5% CO2, cell monolayers were washed with saline, fixed with 100% methanol, and stained with 10% Giemsa. Colonies, defined as being >50 cells, were counted with the use of a light microscope. The surviving fraction is defined as follows: Surviving fraction = [fraction of viable cells recovered x (cloning efficiency of treated cells/cloning efficiency of untreated, control cells)].
MTT Assay and Drug Interaction Analysis.
SCC cells were plated at 1.5 x 103 cells/well into 96-well tissue culture plates (Corning Glass Inc., Corning, NY) and incubated at 37°C in a humidified atmosphere containing 5% CO2. After a 24-h recovery, cells were either untreated or treated for 24 h with varying doses of calcitriol. Subsequently, cells received no further treatment or were treated for 24 h with varying doses of paclitaxel. Plates were harvested by staining with 0.5% MTT, and the absorbance was read with an ELISA reader (model EL-340; Bio-Tek Instruments, Winooski, VT) at 460 nM. Drug interactions were quantitated by median-dose effect analysis (21)
, and combination index values were derived using CalcuSyn software (Biosoft, Ferguson, MO), as described previously (22)
. CI values of <1, =1, and >1 indicate synergism, additivity, and antagonism between the drugs, respectively.
In Vivo Excision Clonogenic Assay.
Mice with 9-day SCCs (three to five animals/group) were treated with saline or 2.5 µg of calcitriol each day for 3 days. On day 3, mice also received varying doses of paclitaxel. Twenty-four h after the last injection, the animals were sacrificed, and their tumors were excised. Aliquots of minced tumor were enzymatically dissociated for 60 min at room temperature with a mixture of type I collagenase, DNase, and EDTA. For each treatment group, a fixed number of viable tumor cells, as determined by trypan blue staining, were then plated in six-well tissue culture plates. After incubation for 7 days, colonies were counted, and the surviving fraction was calculated using the equation: Surviving fraction = (the cloning efficiency of treated cells/cloning efficiency of untreated, control cells). The surviving fraction per gram of tumor is defined as the number of clonogenic tumor cells per gram of treated tumor divided by the number of clonogenic tumor cells per gram of control, untreated tumor.
Tumor Growth Inhibition.
To examine the in vivo antitumor activity of calcitriol, paclitaxel, or the combination of calcitriol with paclitaxel, treatment was initiated on animals bearing palpable SCC or PC-3 tumors. Animals were treated for 3 days with single, daily i.p. injections of saline or calcitriol. On day 3, animals also received a single i.p. injection of paclitaxel. Tumor measurements were obtained using calipers prior to initiating treatment (initial tumor volume) and on the days indicated. Tumor volumes were calculated by the following formula: volume = (length x width2)/2. For each tumor, fractional tumor volumes were calculated using the following formula: Fractional tumor volume = (volume on day measured)/(initial tumor volume).
Preparation of Cell Lysates and Western Blot Analysis.
PC-3 cells were seeded into T75 flasks at densities of 1 x 104 to 2 x 104 cells/ml. Forty-eight h later, the medium was replaced. Treatments were done by adding concentrated drug stocks directly to the culture media. At various times, cells that detached from the tissue culture plate were harvested by collecting the culture medium. After removing these cells, adherent cells were scraped up into PBS. Detached and adherent cells were maintained as separate populations. Cells were collected by centrifugation and washed once in PBS, and the resulting pellets were stored at -70°C. Protein extracts were prepared by resuspending cell pellets in lysis buffer [1% Triton X-100, 0.1% SDS, 50 mM Tris, and 150 mM NaCl, containing 1x protease inhibitor cocktail (PharMingen)] for 30 min on ice. Lysates were transferred to 1.5-ml Eppendorf tubes and clarified by centrifugation at 13,000 rpm for 10 min at 4°C. Proteins were quantitated in duplicate using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) according to manufacturer’s directions. Protein lysates were stored at -70°C until use.
Proteins were resolved on SDS-polyacrylamide gels under denaturing conditions and then electrophoretically transferred to PVDF membranes (NEN Life Science Products, Boston, MA) overnight at 4°C. At room temperature, membranes were blocked for a minimum of 1 h in a 5% w/v solution of nonfat milk in TBST (10 mM Tris, pH 7.6, 150 mM NaCl, and 0.05% Tween 20) and then incubated for 1 h with primary antibody. The blots were washed three times in TBST and subsequently incubated with secondary antibody conjugated with horseradish peroxidase for 1 h. The blots were again washed, and the proteins were detected using Renaissance Western blot chemiluminescence reagents (NEN Life Science Products).
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RESULTS |
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; Ref. 7
). Paclitaxel alone also inhibits SCC survival, with an IC50 of 23 nM. Significantly greater antitumor activity was achieved when calcitriol was combined with paclitaxel (Fig. 1)
. Calcitriol pretreatment potentiated paclitaxel activity to a greater extent than concurrent treatment, except at the highest dose of paclitaxel studied. These results demonstrate that antitumor activity in SCC is increased by combining calcitriol with paclitaxel, and the optimal schedule for administration is treatment with calcitriol, followed by paclitaxel.
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, calcitriol alone had detectable antiproliferative activity in these cells, with an IC50 of 5 µM. Paclitaxel, when used as a single agent, reduced PC-3 clonogenic survival in a concentration-dependent manner. Significantly greater growth inhibition was achieved by pretreating the cells with calcitriol at each of the paclitaxel concentrations tested. Therefore, the combination of calcitriol plus paclitaxel displays increased antiproliferative activity in murine SCC and human prostatic adenocarcinoma, indicating that the effects are not cell type specific.
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, CIs <1.0 were obtained for all combinations of calcitriol and paclitaxel examined, indicating that the interaction between the two drugs is synergistic.
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, the combination of calcitriol plus paclitaxel resulted in a significantly greater decrease in surviving fraction as compared with paclitaxel or calcitriol alone. Thus, greater in vivo antitumor activity is achieved in SCC by pretreatment with calcitriol followed by paclitaxel.
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. In contrast, the combination of calcitriol with paclitaxel resulted in significant tumor regression (Fig. 4B)
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To determine whether calcitriol plus paclitaxel combination therapy also displays increased antiproliferative activity in vivo in PC-3, tumor-bearing mice were treated with saline, calcitriol or paclitaxel alone, or calcitriol in combination with paclitaxel using the dosing schedule described for SCC. As shown in Fig. 5A
, neither paclitaxel nor calcitriol had significant activity when used as a single-agent therapy in PC-3. However, significant antitumor activity was observed when PC-3 tumor-bearing mice were treated with calcitriol followed by paclitaxel (Fig. 5A)
. In this model, nearly complete inhibition of tumor growth was maintained for >2 weeks.
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). These data indicate that previously treated tumors remain responsive to calcitriol plus paclitaxel and that prolonged antitumor activity may be achieved by repeated cycles of therapy.
Calcitriol Decreases p21 Expression and Accelerates Paclitaxel-induced Apoptosis.
Given the findings that loss of p21 sensitizes MCF-7 and HCT116 cells to paclitaxel (17
, 18)
and calcitriol decreases p21 expression in SCC (14)
, we hypothesized that calcitriol enhances paclitaxel antitumor activity via its effects on p21. To test whether calcitriol treatment decreases p21 expression in PC-3 as it does in SCC, cells were treated in vitro with ethanol vehicle control or calcitriol. At various times, whole cell lysates were prepared and analyzed for p21 expression by Western blot. As shown in Fig. 6
, calcitriol treatment resulted in a 60% decrease in p21 expression in PC-3 cells after 72 h and an 80% decrease in expression after 96 h.
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. Cells subsequently received no further treatment or were treated with paclitaxel alone or calcitriol plus paclitaxel. Viable cells were counted 24 h after paclitaxel addition. Coadministration of calcitriol plus paclitaxel resulted in a greater reduction in cell number than treatment with either agent alone (Table 1)
. However, the effect of the combination was only additive. In contrast, a greater than additive reduction in cell number was obtained when cells were pretreated with calcitriol followed by paclitaxel. Thus, a calcitriol pretreatment that reduces p21 expression further enhances paclitaxel cytotoxicity in PC-3.
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, PC-3 cells were either treated with ethanol control or pretreated with 5 µM calcitriol for 72 h, a time sufficient for p21 down-modulation. Subsequently, cells received no further treatment, were treated with paclitaxel alone, or were treated with calcitriol plus paclitaxel for varying lengths of time. Inspection of treated cells revealed two morphologically distinct populations, one cell population remained adherent after treatment whereas a second population detached from the culture dishes. These populations were analyzed separately.
At 24 h, paclitaxel induced Bcl-2 phosphorylation in both the adherent and detached cell populations (Fig. 7)
. Whereas unphosphorylated Bcl-2 was most abundant in the paclitaxel-treated adherent cells, only the phosphorylated forms of Bcl-2 were detected in the detached cells. Phosphorylated Bcl-2 species were still detected in both cell populations after 48 h of treatment but were virtually absent by 72 h. Calcitriol had little effect on Bcl-2, and neither pretreatment nor concurrent treatment with calcitriol altered the effects of paclitaxel on Bcl-2 expression/phosphorylation.
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. At this time, paclitaxel treatment resulted in a 50% reduction in PARP. Although calcitriol itself did not induce apoptosis, it enhanced the effects of paclitaxel such that cells treated with calcitriol prior to or in combination with paclitaxel displayed a 78% reduction in PARP. By 72 h, PARP was no longer detected in the cells that detached after paclitaxel administration, and addition of calcitriol had no further discernible effect. Calcitriol did not alter the effects of paclitaxel on PARP expression in the adherent cell population at any of the times examined. The data obtained at 48 h indicate that calcitriol can accelerate paclitaxel-induced apoptosis in a subset of PC-3 cells in a schedule-independent manner. |
DISCUSSION |
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Preclinically, we demonstrated that there is an increase in antitumor activity in prostatic adenocarcinoma using calcitriol in combination with paclitaxel in vitro and in vivo as measured in clonogenic assays and tumor growth inhibition studies. On the basis of these findings, we propose that calcitriol plus paclitaxel combination therapy may have utility in the treatment of patients with prostate cancer.
The clinical use of calcitriol may be restricted by its dose-limiting toxicity, hypercalcemia. However, a variety of calcitriol analogues, including ILX-23–7553 and EB1089, have been described that possess antiproliferative activity in vivo without inducing hypercalcemia (7 , 30) . It has been shown recently that EB1089, when combined with paclitaxel, inhibits the growth of MCF-7 breast cancer cells in vivo (31) . Furthermore, we observed that paclitaxel appears to attenuate calcitriol-mediated hypercalcemia in preclinical models (data not shown). Agents that disrupt or stabilize microtubules can inhibit calcium transport, which may account for this activity (32) . Thus, paclitaxel and either calcitriol or analogues may be a safe and effective combination in the treatment of human cancer.
We have further demonstrated that calcitriol enhances paclitaxel antiproliferative activity in vitro and in vivo in the murine SCC model, SCCVII/SF. A previous report indicates that these cells are relatively resistant to paclitaxel in vivo at a concentration of 40 mg/kg (33)
. We found that although paclitaxel (20 mg/kg) has little activity when administered to tumor-bearing mice as a single agent, pretreatment with calcitriol yields substantial antitumor activity (Fig. 4B)
. These data suggest that calcitriol and paclitaxel combination therapy may be useful, even in the treatment of tumors that are paclitaxel insensitive.
Paclitaxel cytotoxicity is increased in MCF-7 breast cancer cells and HCT116 colon cancer cells when p21 expression is specifically perturbed (17
, 18)
. Because calcitriol treatment reduces p21 expression in the SCC model (14)
and in PC-3 cells (Fig. 6)
, we hypothesized that calcitriol might enhance the antitumor activity of paclitaxel via its effect on p21. To test this, we examined whether paclitaxel effects were enhanced in cells pretreated with calcitriol (p21 low) as compared with cells receiving concurrent calcitriol (baseline p21) and paclitaxel.
Paclitaxel effects were measured by following changes in viable cell number (Table 1)
and molecular markers (Fig. 7)
. In the cell recovery studies, we observed a schedule dependence such that the greatest reduction in cell number occurs when cells are pretreated with calcitriol for 72 h followed by a 24-h treatment with paclitaxel. Interestingly, the reduction in cell number cannot be accounted for by an increase in apoptosis because no PARP cleavage was detected 24 h after paclitaxel addition (Fig. 7)
. However, a different observation is made 48 h after paclitaxel addition. At this time, calcitriol alone does not induce apoptosis but does enhance the level of PARP cleavage observed in the detached cell population in the presence of paclitaxel. This enhancement is schedule independent, with similar effects observed in paclitaxel-treated cells regardless of whether they were pretreated or concurrently treated with calcitriol.
On the basis of these data, we propose a model in which the "early" (within 24 h) enhancement of paclitaxel cytotoxicity by calcitriol is schedule dependent and is not attributed to acceleration of paclitaxel-induced apoptosis. The schedule dependence may reflect the time required for calcitriol treatment to decrease p21 expression. The "delayed" (at 48 h) enhancement of paclitaxel activity by calcitriol is schedule independent and associated with acceleration of apoptosis in a subset of PC-3 cells. Recent work from our laboratory demonstrates that calcitriol inhibits specific survival signals in cells that detach during treatment.4 Such inhibition may render these cells more susceptible to the proapoptotic signals generated by paclitaxel. The ability of calcitriol to reduce cell survival signals may also explain how it can enhance the antitumor activity of mechanistically diverse cytotoxic agents, such as cisplatin (13) and paclitaxel. Studies to address these and related issues are in progress.
Paclitaxel-mediated apoptosis in LNCaP and PC-3 prostate cancer cells has been associated with Bcl-2 phosphorylation and inactivation (23) and/or down-modulation of the related apoptotic suppressor, Bcl-XL (34) . Consistent with these results, we found that within 24 h, paclitaxel treatment resulted in phosphorylation of the apoptotic suppressor protein, Bcl-2. Loss or inactivation of Bcl-2 in prostate cancer cells after paclitaxel administration has been proposed to promote cell death by shifting the intracellular balance of death regulators in favor of proapoptotic molecules such as Bax (23) . In our studies, paclitaxel-mediated changes in the intracellular levels of Bcl-2 temporally precede the loss of full-length PARP, suggesting that they may initiate the apoptotic program.
Wang et al. (35) demonstrated recently that calcitriol pretreatment increases paclitaxel induction of cell death and paclitaxel antitumor activity in vitro in MCF-7 breast cancer cells. However, in contrast to our findings, calcitriol modestly increased the effect of paclitaxel on Bcl-2 phosphorylation. Comparison of these two studies reveals that although a 24-h exposure to 100 nM paclitaxel results in minimal Bcl-2 phosphorylation in MCF-7, this exposure results in strong induction of Bcl-2 phosphorylation in PC-3. This strong induction may have prohibited the detection of a subtle effect of calcitriol on paclitaxel-mediated Bcl-2 phosphorylation in PC-3.
In summary, our data demonstrate that calcitriol enhances paclitaxel antitumor activity in PC-3 and SCC cells in vitro and in vivo and indicate that novel calcitriol/paclitaxel-based combination therapies may have significant clinical utility in the treatment of a variety of solid tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by USPHS Grant CA67267 from the National Cancer Institute, NIH; United States Army Medical Research and Material Command Grant DAMD 17-98-1-8549; and grants from CaPCURE and the Mary Hillman Jennings Foundation. 
2 To whom requests for reprints should be addressed, at University of Pittsburgh, Department of Pharmacology, W1002 Biomedical Science Tower, Pittsburgh, PA 15213. Phone: (412) 648-2344; Fax: (412) 624-7737; E-mail: pah13+{at}pitt.edu
3 The abbreviations used are: cdk, cyclin-dependent kinase; SCC, squamous cell carcinoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; CI, combination index; PSA, prostate-specific antigen; PVDF, polyvinylidene difluoride.
4 T. F. McGuire, D. L. Trump, and C. S. Johnson. Vitamin D3-induced apoptosis of murine squamous cell carcinoma cells: selective induction of caspase-dependent MEK cleavage and up-regulation of MEKK1, submitted for publication.
Received 10/ 3/00; revised 1/18/01; accepted 1/19/01.
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P. A. Hershberger, T. F. McGuire, W.-D. Yu, E. G. Zuhowski, J. H. M. Schellens, M. J. Egorin, D. L. Trump, and C. S. Johnson Cisplatin Potentiates 1,25-Dihydroxyv |
), with varying doses of paclitaxel alone (
), pretreated with calcitriol followed by paclitaxel (•), or treated simultaneously with calcitriol plus paclitaxel (
), as measured by growth inhibition in the 7-day in vitro clonogenic assay. In pretreated cells, calcitriol was added 24 h prior to paclitaxel. Cells were exposed to paclitaxel for a total of 24 h. Calcitriol was used at a concentration of 4 nM. Each point represents the mean surviving fraction as determined by counting triplicate wells; bars, 95% confidence intervals. Note that the symbol for the calcitriol-alone treatment group is arbitrarily placed with respect to the X axis. Values for pretreatment with calcitriol followed by paclitaxel are significantly different from those obtained for paclitaxel alone; *, P 
0.01.
), 1.25 µg of calcitriol daily for 3 days (
