Pamela A. Hershberger

Anti-tumor activity of calcitriol: pre-clinical and clinical studies.

1,25-Dihydroxycholecalciferol (calcitriol) is recognized widely for its effects on bone and mineral metabolism. Epidemiological data suggest that low Vitamin D levels may play a role in the genesis of prostate cancer and perhaps other tumors. Calcitriol is a potent anti-proliferative agent in a wide variety of malignant cell types. In prostate, breast, colorectal, head/neck and lung cancer as well as lymphoma, leukemia and myeloma model systems calcitriol has significant anti-tumor activity in vitro and in vivo. Calcitriol effects are associated with an increase in G0/G1 arrest, induction of apoptosis and differentiation, modulation of expression of growth factor receptors. Glucocorticoids potentiate the anti-tumor effect of calcitriol and decrease calcitriol-induced hypercalcemia. Calcitriol potentiates the antitumor effects of many cytotoxic agents and inhibits motility and invasiveness of tumor cells and formation of new blood vessels. Phase I and II trials of calcitriol either alone or in combination with carboplatin, taxanes or dexamethasone have been initiated in patients with androgen dependent and independent prostate cancer and advanced cancer. Data indicate that high-dose calcitriol is feasible on an intermittent schedule, no dose-limiting toxicity has been encountered and optimal dose and schedule are being delineated. Clinical responses have been seen with the combination of high dose calcitriol+dexamethasone in androgen independent prostate cancer (AIPC) and apparent potentiation of the antitumor effects of docetaxel have been seen in AIPC. These results demonstrate that high intermittent doses of calcitriol can be administered to patients without toxicity, that the MTD is yet to be determined and that calcitriol has potential as an anti-cancer agent.

Regulation of Endogenous Gene Expression in Human Non–Small Cell Lung Cancer Cells by Estrogen Receptor Ligands

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non-small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERalpha, ERbeta, and ER coregulators in human lung fibroblasts and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERbeta and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERalpha or the p160 coactivator SRC-1. ERbeta and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist 17-beta-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal growth factor, which served as a positive control for an alternative growth stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal growth factor also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

Pharmacokinetics of high-dose oral calcitriol: results from a phase 1 trial of calcitriol and paclitaxel.

The data reported are from a trial designed to determine, in patients with advanced cancer, the maximum tolerated dose and pharmacokinetics of calcitriol when administered with paclitaxel, an agent whose antitumor activity in in vitro and in vivo studies has been shown to be enhanced by calcitriol. An additional goal was to evaluate the relationship between calcitriol dose and hypercalcemia.

EFFECTS OF VITAMIN D (CALCITRIOL) ON TRANSITIONAL CELL CARCINOMA OF THE BLADDER IN VITRO AND IN VIVO

PURPOSE Vitamin D (calcitriol) has significant antiproliferative effects on various tumor cells in vitro and in vivo. In the clinical situation a major impediment to systemic administration of calcitriol is the side effect of hypercalcemia. To test the potential usefulness of calcitriol for bladder cancer treatment, we studied the antiproliferative effect of vitamin D on 2 human bladder cancer cell lines, 253j and T-24, in vitro. We also examined the in vivo effects of calcitriol in an animal model of bladder cancer using intravesical administration to avoid the toxicity of systemic calcitriol therapy. MATERIALS AND METHODS The presence of vitamin D receptors in normal and neoplastic human bladder tissue, and tumor cells T-24 and 253j was determined by immunoblot analysis. Tumor cell proliferation in the presence or absence of calcitriol was determined using a crystal violet assay. Calcitriol induced apoptosis was determined by morphology, polyadenosine diphosphate ribose polymerase cleavage and annexin V binding. In vivo studies were performed by weekly intravesical instillation of calcitriol in female Fischer 344 rats after induction of tumors by N-methyl nitrosourea. Calcitriol administration was started 3 weeks after tumor induction for 7 doses at weekly intervals. RESULTS Normal and neoplastic human bladder tissue, and the cell lines expressed vitamin D receptors. In the 253j and T-24 cell lines proliferation was significantly inhibited by calcitriol. Progressive cleavage of full length polyadenosine diphosphate ribose polymerase was observed in calcitriol treated cells starting as early as 4 hours after exposure. Similar changes were not observed in the control cells treated with vehicle (ethanol) alone. After 24 hours of treatment with calcitriol 45.8% of 253j cells bound annexin compared to 16.5% of control cells (chi-square p <0.001). Of the control animals 66% developed bladder tumors and 55% of the animals treated with calcitriol early (3 weeks) after tumor induction developed bladder tumors. Almost all of the tumors that developed in the calcitriol group were unifocal, and only 20% were invasive compared to 50% of those in the control animals. CONCLUSIONS These results demonstrate that calcitriol inhibits proliferation and induces apoptosis in human bladder tumor cells in vitro, and may have therapeutic potential in bladder cancer. In vivo studies using an N-methylnitrosourea induced model of bladder cancer demonstrate that early institution of intravesical calcitriol therapy after carcinogen exposure results in fewer tumors, which are also less likely to be multifocal, high grade or invasive. With our protocol a short course of intravesical calcitriol administration did not result in any significant toxicity.

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

1α,25‐Dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25‐hydroxyvitamin D3‐24‐hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT‐PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor‐specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient‐matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) assay for 1,25D3 and its CYP24‐generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time‐dependent loss of 1,25D3 coincident with the appearance of CYP24‐generated metabolites. MK‐24(S)‐S(O)(NH)‐Ph‐1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half‐life. Furthermore, combination of 1,25D3 with MK‐24(S)‐S(O)(NH)‐Ph‐1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

Estrogen Receptor Signaling in Lung Cancer

Lung cancer has long been thought of as a cancer that mainly affects men, but over the past several decades, because of the high increase in tobacco use by women, there has been a corresponding dramatic increase in lung cancer among women. Since 1998, lung cancer deaths in women have surpassed those caused by breast cancer in the United States. Annual lung cancer deaths among US women currently surpass those caused by breast, ovarian, and cervical cancers combined. Women are more likely than men to be diagnosed with adenocarcinoma and small cell carcinoma of the lung compared to squamous cell carcinoma, and never-smokers diagnosed with lung cancer are almost three times more likely to be female than male. These observations in the population, coupled to the findings that both estrogen receptors (ERs) and aromatase, the enzyme that synthesizes 17beta-estradiol, are expressed by lung tumors, suggest a role for female steroid hormones in control of lung cancer growth. Preclinical data and clinical data are increasingly emerging to support this concept, and to suggest that a local production of estrogen and expression of ERs occurs in lung tumors that arise in men as well as in women. An additional protein that recognizes 17beta-estradiol with high affinity, GPR30, also is expressed in lung tumors at high levels and may be responsible for some of the proliferation signals induced by estrogen.

The Candidate Oncogene CYP24A1: A Potential Biomarker for Colorectal Tumorigenesis

The main autocrine/paracrine role of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25-D3), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D3, we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D3 availability, decreasing its antiproliferative effect.

Vitamin D-related therapies in prostate cancer.

Calcitriol or 1,25-dihydroxycholecalciferol (vitamin D) is classically known for its effects on bone and mineral metabolism. Epidemiological data suggest that low vitamin D levels increase the risk and mortality from prostate cancer. Calcitriol is also a potent anti-proliferative agent in a wide variety of malignant cell types including prostate cancer cells. In prostate model systems (PC-3, LNCaP, DU145, MLL) calcitriol has significant anti-tumor activity in vitro and in vivo. Calcitriols effects are associated with an increase in cell cycle arrest, apoptosis, differentiation and in the modulation of growth factor receptors. Calcitriol induces a significant G0/G1 arrest and modulates p21Waf1/Cip1 and p27Kip1, the cyclin dependent kinase inhibitors. Calcitriol induces PARP cleavage, increases the bax/bcl-2 ratio, reduces levels of phosphorylated mitogen-activated protein kinases (P-MAPKs, P-Erk-1/2) and phosphorylated Akt (P-Akt), induces caspase-dependent MEK cleavage and up-regulation of MEKK-1, all potential markers of the apoptotic pathway. Glucocorticoids potentiate the anti-tumor effect of calcitriol and decrease calcitriol-induced hypercalcemia. In combination with calcitriol, dexamethasone results in a significant time- and dose-dependent increase in VDR protein and an enhanced apoptotic response as compared to calcitriol alone. Calcitriol can also significantly increase cytotoxic drug-mediated anti-tumor efficacy. As a result, phase I and II trials of calcitriol either alone or in combination with the carboplatin, paclitaxel, or dexamethasone have been initiated in patients with androgen-dependent and -independent prostate cancer and advanced cancer. Patients were evaluated for toxicity, maximum tolerated dose (MTD), schedule effects, and PSA response. Data from these studies indicate that high-dose calcitriol is feasible on an intermittent schedule, the MTD is still being delineated and dexamethasone or paclitaxel appear to ameliorate toxicity. Studies continue to define the MTD of calcitriol which can be safely administered on this intermittent schedule either alone or with other agents and to evaluate the mechanisms of calcitriol effects in prostate cancer.

Vorinostat increases carboplatin and paclitaxel activity in non-small cell lung cancer cells

We observed a 53% response rate in non‐small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 μM) inhibited growth by: 17% ± 7% in A549, 28% ± 6% in 128‐88T, 39% ± 8% in Calu1 and 41% ± 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 μM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128‐88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 μM vorinostat, 83% ± 10%; 5 μM carboplatin, 41% ± 11%; carboplatin/vorinostat, 8% ± 4%; 2 nM paclitaxel, 53% ± 11%; paclitaxel/vorinostat, 46% ± 21%. In A549 and 128‐88T, vorinostat potentiated carboplatin induction of gamma‐H2AX (a DNA damage marker) and increased α‐tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in α‐tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat‐mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.

Flavourings significantly affect inhalation toxicity of aerosol generated from electronic nicotine delivery systems (ENDS)

Background E-cigarettes or electronic nicotine delivery systems (ENDS) are designed to deliver nicotine-containing aerosol via inhalation. Little is known about the health effects of flavoured ENDS aerosol when inhaled. Methods Aerosol from ENDS was generated using a smoking machine. Various types of ENDS devices or a tank system prefilled with liquids of different flavours, nicotine carrier, variable nicotine concentrations and with modified battery output voltage were tested. A convenience sample of commercial fluids with flavour names of tobacco, piña colada, menthol, coffee and strawberry were used. Flavouring chemicals were identified using gas chromatography/mass spectrometry. H292 human bronchial epithelial cells were directly exposed to 55 puffs of freshly generated ENDS aerosol, tobacco smoke or air (controls) using an air–liquid interface system and the Health Canada intense smoking protocol. The following in vitro toxicological effects were assessed: (1) cell viability, (2) metabolic activity and (3) release of inflammatory mediators (cytokines). Results Exposure to ENDS aerosol resulted in decreased metabolic activity and cell viability and increased release of interleukin (IL)-1β, IL-6, IL-10, CXCL1, CXCL2 and CXCL10 compared to air controls. Cell viability and metabolic activity were more adversely affected by conventional cigarettes than most tested ENDS products. Product type, battery output voltage and flavours significantly affected toxicity of ENDS aerosol, with a strawberry-flavoured product being the most cytotoxic. Conclusions Our data suggest that characteristics of ENDS products, including flavours, may induce inhalation toxicity. Therefore, ENDS users should use the products with caution until more comprehensive studies are performed.