Inhibition of human cancer cell growth by 1,25-dihydroxyvitamin D3 metabolites

Specific high-affinity 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors, which can undergo hormone-dependent activation and nuclear localization, have been demonstrated in a wide variety of established human cancer cell lines and surgically obtained human cancer tissues. 1,25-(OH)2D3 has been reported by some workers to stimulate cancer cell replication at low "physiological" concentrations and by ourselves and others to inhibit at higher concentrations. We report here that 1,25-(OH)2D3 had a biphasic effect on the replication of two distinct human cancer cell lines, i.e., the breast cancer T-47D and the malignant melanoma MM96, an effect analogous to that of estrogens on the breast cancer cell line MCF-7. These inhibitory effects were accompanied by marked morphological changes. Furthermore, two known metabolites of 1,25-(OH)2D3, i.e., 1,24,25-trihydroxyvitamin D3 and 1,25,26-trihydroxyvitamin D3, which compete for binding to the 1,25-(OH)2D3 receptor, did not stimulate but were almost equipotent with 1,25-(OH)2D3 in inhibiting the replication of both cell lines. The stimulatory but not the inhibitory effect of 1,25-(OH)2D3 was abolished by cortisone. These 1,25-dihydroxyvitamin D3 metabolites show promise for the inhibition of cancer growth, analogous to the effect of estrogens and antiestrogens in breast cancer but with potential application in a much wider range of human cancers.     

Influence of tumor derived fibroblasts and 1,25-dihydroxyvitamin D3 on growth of breast cancer cell lines

Summary Fibroblasts are known to be present in variable amounts in human breast adenocarcinoma tissue. In order to investigate if they influence in some way the proliferation rate of the carcinoma cells, we developed a coculture model in which cells of well characterized breast epithelial cell lines were seeded and grown in microchamber slides along with fibroblasts derived from breast tumor biopsies. As representatives of hormone dependent and independent tumor cells, we used MCF-7 and BT-20 cell lines. A third line, NPM-21T, derived from non proliferating mastopathy cells immortalized by SV-40 T DNA transfection, was representative of non tumor epithelial cells. The proliferation rate of the adenocarcinoma and epithelial cells was assessed by measurement of the BrdU labeling index, the cells being identified by specific -actin immunostaining. It was found that the proliferation of the adenocarcinoma cells was significantly increased in the presence of fibroblasts, while that of immortalized cells was not. Moreover, 1,25(OH)₂D₃, which was known to be a negative regulator of carcinoma cell growth, was found to be able also to blunt the overgrowth in the presence of fibroblasts. The absence of response of NPM-21T cells to the presence of fibroblasts suggests that the tumor cells could be the origin of their own overgrowth, through an indirect mechanism mediated by the fibroblasts. The factors which are involved and the 1,25(OH)₂D₃ mechanism of action are not yet identified.Abbreviations 1,25(OH)₂D₃ 1,25-dihydroxycholecalciferol - BrdU 5-bromo-2'-deoxyuridine - LI labeling index - ER, PGR, VDR estrogen, progesterone, and 1,25(OH)₂D₃ receptors - EGF, EGF-R epidermal growth factor and epidermal growth factor receptor     

Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2-mediated JAK/STAT3 signaling in endothelial cell

Tumor angiogenesis is one of the hallmarks of the development in malignant neoplasias and metastasis. Many angiogenesis inhibitors are small molecules from natural products. Indirubin, the active component of a traditional Chinese herbal medicine, Banlangen, has been shown to exhibit antitumor and anti-inflammation effects. But its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is unknown. Here, we identified that indirubin inhibited prostate tumor growth through inhibiting tumor angiogenesis. Using chick chorioallantoic membrane (CAM) assay and mouse corneal model, we found that indirubin inhibited angiogenesis in vivo. We also showed the inhibition activity of indirubin in endothelial cell migration, tube formation and cell survival in vitro. Furthermore, indirubin suppressed vascular endothelial growth factor receptor 2-mediated Janus kinase (JAK)/STAT3 signaling pathway but had little effects on the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in endothelial cell. Our study provided the first evidence for antitumor angiogenesis activity of indirubin and the related molecular mechanism. Our investigations suggested that indirubin was a potential drug candidate for angiogenesis related diseases.     

1,25(OH)2D3 modulation of mammary tumor cell growth in vitro and in vivo

Summary The biological role of 1,25(OH)₂D₃ in controlling Ca⁺⁺ homeostasis in the body has been identified and widely investigated for a long time. More recently its effect in regulating cell proliferation or differentiated activity was described in a variety of normal and malignant cells. The present study was carried out to investigate the different aspects and biological mechanisms of this activity and to determine if the use of 1,25(OH)₂D₃ in the treatment of breast cancer patients could be considered.It is found that 1,25(OH)₂D₃ reduces the proliferation of MCF-7 and BT-20 cell lines regardless of their sex steroid receptor status. This effect is related to the concentration, from 10^–12 M to 10^–8 M. Its amplitude is less in other cell lines, but it opposes the EGF-induced increase of proliferation. It is observed that the proliferation rate of MCF-7 and BT-20 cells is increased when these tumor cells are cocultured with fibroblasts derived from breast tumor biopsies and that 1,25(OH)₂D₃ reverses this process. Moreover, experiments on DMBA induced mammary tumors in Sprague Dawley rats found that 1,25(OH)₂D₃ given at non toxic doses reduces significantly the tumor proliferation.These data showed that 1,25(OH)₂D₃ at low doses is effective on the proliferation of BT-20 and MCF-7 cells and on the paracrine growth stimulatory effect observed in the presence of fibroblasts. They suggest that 1,25(OH)₂D₃ or related synthetic molecules which are less active on Ca⁺⁺ metabolism could be useful in the treatment of breast cancer patients.a Abbreviations used are: 1,25(OH)₂D₃, 1,25-dihydroxycholecalciferol; EGF and EGF-R, epidermal growth factor and epidermal growth factor receptor; BrdU, 5-bromo-2'-deoxyuridine; DMBA, 7,12-dimethylbenz(a)anthracene; VDR, 1,25(OH)₂D₃ receptor; FCS, fetal calf serum.b This investigation has been supported by INSERM grant CRE 9004-14, ARC grant (1992), and the Comité Départemental de Haute-Savoie de la Ligue Nationale Française contre le Cancer.     

An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling

The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1β-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [(125)I]-labeled NRG-1β to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.     

Suppression of in vivo growth of human cancer solid tumor xenografts by 1,25-dihydroxyvitamin D3

It has been demonstrated previously that several human cancer cell lines possess specific, high affinity receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3, calcitriol] and that 1,25-(OH)2D3 and certain of its metabolites inhibit the growth in vitro of several human breast cancer and malignant melanoma cell lines, i.e., analogous to the effect of estrogens on breast cancer. Furthermore, it has been shown that 1,25-(OH)2D3 and one of its synthetic analogues prolonged the survival in mouse leukemia, induced by inoculation of leukemic cells into syngeneic mice. However, until now no growth-inhibitory effect of 1,25-(OH)2D3 has been demonstrated in vivo for human cancer cells or for solid cancers. This paper describes the suppression by 1,25-(OH)2D3 of the growth of human cancer cell-derived xenografts in immune-suppressed mice. However, the 24-hydroxylated metabolite and the 24-difluorinated analogue of 1,25-(OH)2D3, both of which are active in vitro, were ineffective in this xenograft model system. The suppression by 1,25-(OH)2D3, which was achieved without significant toxicity, was observed with xenografts derived from two 1,25-(OH)2D3 receptor-positive cell lines (COLO 206F, derived from a colonic cancer, and COLO 239F from a malignant melanoma) but not in those from a receptor-negative line (RPMI 7932, also derived from a malignant melanoma). These studies demonstrate that pharmacological doses of 1,25-(OH)2D3 can be tolerated in the presence of a low calcium diet and that these doses can suppress the growth of human solid xenograft tumors in vivo. This is the first report of 1,25-(OH)2D3 growth suppression of solid tumors derived from human cancer cells in an in vivo model system, and it supports the hypothesized potential of the hormone in the treatment of 1,25-(OH)2D3 receptor-positive human cancers.     

DSCP1, a novel TP53-inducible gene, is upregulated by strong genotoxic stresses and its overexpression inhibits tumor cell growth in vitro

TP53-inducible genes play crucial roles from many biological aspects including cell cycle control, DNA repair, and apoptosis. Herein we report the identification and characterization of a novel TP53-inducible gene, DSCP1 (damage stimulated cytoplasmic protein 1), localized at 17q11. The gene was expressed ubiquitously in normal adult tissues; its protein product was localized mainly in the cytoplasm with anchoring on unknown subcellular structures. Exogenous expression of TP53 induced expression of DSCP1, but more interestingly, DSCP1 was induced by strong genotoxic stresses not only in TP53-maintaining cells but also in TP53-dysfunctioning cells, although the induction was much more efficient in the former than in the latter. In cultured cancer cells, the basal expression level appeared to depend on the functional status of TP53. Moreover, exogenous overexpression of DSCP1 retarded cancer cell growth in vitro. These results indicate that DSCP1 is a stress-inducible gene in both a TP53 dependent and independent manner and that its protein product can inhibit cancer cell growth.     

Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid.

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.     

Protection against cyclophosphamide-induced alopecia and inhibition of mammary tumor growth by topical 1,25-dihydroxyvitamin D3 in mice

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)₂D₃ was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)₂D₃, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)₂D₃-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)₂D₃ alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)₂D₃ pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)₂D₃ can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)₂D₃ was shown, either alone or in combination with CTX, to inhibit tumor growth. Int. J. Cancer 75:303–309, 1998. © 1998 Wiley-Liss, Inc.     

Serum deprivation response inhibits breast cancer progression by blocking transforming growth factor‐β signaling

Serum deprivation response (SDPR), a key substrate for protein kinase C, play a critical role in inducing membrane curvature and participate in the formation of caveolae. However, the function of SDPR in cancer development and progression is still not clear. Here, we found that SDPR is downregulated in human breast cancer. Overexpression of SDPR suppresses cell proliferation and invasion in MDA‐MB‐231 cells, while depletion of SDPR promotes cell proliferation and invasion in MCF10A cells. Subsequently, SDPR depletion induces epithelial–mesenchymal transition (EMT)‐like phenotype. Finally, knockdown of SDPR activates transforming growth factor‐β (TGF‐β) signaling by upregulation of TGF‐β1 expression. In conclusion, our results showed that SDPR inhibits breast cancer progression by blocking TGF‐β signaling. Serum deprivation response suppresses cell proliferation and invasion in breast cancer cells. SDPR depletion induces epithelial–mesenchymal transition by activation of TGF‐β signaling.     

Prostate tumor progression is mediated by a paracrine TGF-beta/Wnt3a signaling axis

Transforming growth factor (TGF)-beta is an important paracrine factor in tumorigenesis. Ligand binding of the type I and II TGF-beta receptors initiate downstream signaling. The role of stromal TGF-beta signaling in prostate cancer progression is unknown. In mice, the conditional stromal knockout of the TGF-beta type II receptor expression (Tgfbr2(fspKO)) resulted in the development of prostatic intraepithelial neoplasia and progression to adenocarcinoma within 7 months. Clinically, we observed a loss of TGF-beta receptor type II expression in 69% of human prostate cancer-associated stroma, compared to 15% of stroma associated with benign tissues (n=140, P-value <0.0001). To investigate the mechanism of paracrine TGF-beta signaling in prostate cancer progression, we compared the effect of the prostatic stromal cells from Tgfbr2(fspKO) and floxed TGF-beta type II receptor Tgfbr2(floxE2/floxE2) mice on LNCaP human prostate cancer cells in vitro and tissue recombination xenografts. Induction of LNCaP cell proliferation and tumorigenesis was observed by Tgfbr2(fspKO) prostate stroma as a result of elevated Wnt3a expression. Neutralizing antibodies to Wnt3a reversed LNCaP tumorigenesis. The TGF-beta inhibition of Wnt3a expression was in part through the suppression of Stat3 activity on the Wnt3a promoter. In conclusion, the frequent loss of stromal TGF-beta type II receptor expression in human prostate cancer can relieve the paracrine suppression of Wnt3a expression.     

Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model

Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. Here, we use a Drosophila tumor model to identify genes, which are specifically required for tumor growth. We found that reduction of phosphoinositide 3-kinase (PI3K) activity resulted in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. We conclude that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results presented here broaden our insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression.     

DCUN1D3, a novel UVC-responsive gene that is involved in cell cycle progression and cell growth

DCUN1D3 (DCN1, defective in cullin neddylation 1, domain containing 3) was found during the process of high throughput screening of novel human genes associated with serum response element (SRE) pathway activation. The DCUN1D3 gene is highly conserved among vertebrates. Human DCUN1D3 complementary DNA (cDNA) encodes 304 amino acids with an apparent molecular mass of 34 kDa. However, there has been no report about the function of DCUN1D3. This study detected that DCUN1D3 was broadly expressed in several tumor tissues and cultured cell lines; however, UVC irradiation of different doses significantly increased DCUN1D3 expression level in these cancer cell lines. Over-expression of the DCUN1D3 inhibits cell growth in HeLa. When the DCUN1D3 gene was silenced by siRNA in UVC-treated HeLa, the cell cycle in S phase was remarkably blocked; furthermore, the UVC-induced cell death was inhibited. In addition, DCUN1D3 localized mainly in the cytoplasm and perinuclear, but after UVC treatment, the DCUN1D3 gradually entered the nucleus. All the results above indicate that DCUN1D3 is a novel UVC-response gene involved in cell cycle regulation and cell survival.     

A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin

Both vascular endothelial growth factor A (VEGF) and osteopontin (OPN) can directly induce tumor angiogenesis, which is essential for the growth and metastasis of solid tumors. Here we engineered a bispecific antibody (VEGF/OPN-BsAb) using the anti-VEGF-A antibody bevacizumab and the anti-OPN antibody hu1A12. Compared with hu1A12 alone and bevacizumab alone, VEGF/OPN-BsAb was significantly more effective in inhibiting tumor angiogenesis in a highly metastatic human hepatocellular carcinoma nude mouse model. Further study demonstrated that VEGF/OPN-BsAb could effectively suppress primary tumor growth and metastasis to lungs, suggesting that it might be a promising therapeutic agent for treatment of metastatic cancer.     

1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and down-modulates p21(Waf1/Cip1) in vitro and in vivo.

1,25-Dihydroxycholecalciferol (1,25-D3) has significant antitumor effects in the murine squamous cell carcinoma (SCC) tumor model in vitro and in vivo. We investigated the basis for this antiproliferative activity and found that, in vitro, 1,25-D3 administration is associated with altered expression of cell cycle regulatory proteins, treatment results in retinoblastoma dephosphorylation, decreased expression of p21(Waf1/Cip1) (p21) mRNA and protein, and increased expression of p27Kip1 (p27) mRNA and protein. Dexamethasone, which acts synergistically with 1,25-D3 to inhibit SCC proliferation, enhanced 1,25-D3-induced down-modulation of p21 without affecting the ability of 1,25-D3 to increase p27 expression. 1,25-D3 did not induce cleavage of poly(ADP-ribose) polymerase. These in vitro data suggest that 1,25-D3 exerts antitumor activity in SCC by perturbing cell cycle progression rather than by inducing apoptosis. In vivo, a 1,25-D3 treatment regimen that results in a decrease in SCC tumor volume is associated with a statistically significant decrease in intratumoral p21 expression. p21 expression is not changed in tumors isolated from control animals or animals treated with a nontherapeutic dose of 1,25-D3. Intratumoral p27 levels were not modulated by 1,25-D3 treatment. Thus, both in vitro and in vivo, 1,25-D3-mediated growth inhibition is associated with p21 down-modulation.     

Silibinin inhibits colorectal cancer growth by inhibiting tumor cell proliferation and angiogenesis

Herein, for the first time, we investigated in vivo efficacy and associated molecular biomarkers and mechanisms of a chemopreventive agent, silibinin, against human colorectal carcinoma (CRC) HT29 xenograft growth. Nude mice were implanted with HT29 cells and fed with vehicle (carboxymethyl cellulose or phosphatidylcholine) or 200 mg/kg/d dose of silibinin or 100 and 200 mg/kg/d doses of silybin-phytosome (5 days per week) for 32 days. Silibinin inhibited tumor growth that accounted for 48% (P = 0.002) decrease in tumor volume and 42% (P = 0.012) decrease in tumor weight at the end of the experiment without any adverse health effect. A stronger antitumor efficacy was observed with silybin-phytosome preparation. Silibinin decreased proliferation index by 40% (P < 0.001), increased apoptotic index by approximately 2-fold (P = 0.001), and reduced microvessel density by 36% (P = 0.001) in tumors. Antiproliferative and proapoptotic effects of silibinin were associated with down-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation as well as cyclin D1 expression. Antiangiogenic effect of silibinin was coupled with a strong decrease in inducible nitric oxide synthase (NOS) and NOS3, cyclooxygenase-1 (COX-1) and COX-2, and hypoxia-inducing factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). These findings suggest in vivo antitumor efficacy of silibinin against CRC involving its antiproliferative, proapoptotic, and antiangiogenic activities. The inhibition of ERK1/2 and Akt signaling may account for antiproliferative and proapoptotic effects, whereas down-regulation of NOS, COX, HIF-1 alpha, and VEGF expression could lead to antiangiogenic effect of silibinin against CRC. Overall, potential use of silibinin against human CRC could be suggested.     

Alpha1-antitrypsin inhibits angiogenesis and tumor growth

Disturbances of the ratio between angiogenic inducers and inhibitors in tumor microenvironment are the driving force behind angiogenic switch critical for tumor progression. Angiogenic inhibitors may vary depending on organismal age and the tissue of origin. We showed that alpha(1)-antitrypsin (AAT), a serine protease inhibitor (serpin) is an inhibitor of angiogenesis, which induced apoptosis and inhibited chemotaxis of endothelial cells. S- and Z-type mutations that cause abnormal folding and defective serpin activity abrogated AAT antiangiogenic activity. Removal of the C-terminal reactive site loop had no effect on its angiostatic activity. Both native AAT and AAT truncated on C-terminus (AATDelta) inhibited neovascularization in the rat cornea and delayed the growth of subcutaneous tumors in mice. Treatment with native AAT and truncated AATDelta, but not control vehicle reduced tumor microvessel density, while increasing apoptosis within tumor endothelium. Comparative analysis of the human tumors and normal tissues of origin showed correlation between reduced local alpha(1)-antitrypsin expression and more aggressive tumor growth.