A new human breast cancer cell line, KPL-3C, secretes parathyroid hormone-related protein and produces tumours associated with microcalcifications in nude mice.

Parathyroid hormone-related protein (PTHrP) is the main cause of humoral hypercalcaemia of malignancy (HHM). We recently established a new human breast cancer cell line, designated KPL-3C, from the malignant effusion of a breast cancer patient with HHM. Morphological, cytogenetic and immunohistochemical analyses indicated that the cell line is derived from human breast cancer. The KPL-3C cells stably secrete immunoreactive PTHrP measured by a two-site immunoradiometric assay, possess both oestrogen and progesterone receptors and are tumorigenic in female nude mice. The addition of phorbol-12-myristate-13-acetate to the medium significantly increased PTHrP secretion from the cells. In contrast, hydrocortisone, medroxyprogesterone acetate and 22-oxacalcitriol decreased PTHrP secretion in a dose-dependent manner. Unexpectedly, a number of microcalcifications were observed in the transplanted tumours. Radiographical examination indicated that the microcalcifications in the tumours are very similar to those commonly observed in human breast cancer. These findings suggest that this KPL-3C cell line may be useful for studying the regulatory mechanisms of PTHrP secretion and the mechanisms that lead to the deposition of microcalcifications in breast cancer.     

VEGF EXPRESSION IS INHIBITED BY APIGENIN IN HUMAN BREAST CANCER CELLS

Objective： To study the effects of apigenin on vascular endothelial growth factor （VEGF） in human breast cancer cells （MDA-MB-231. Methods： MTT assay was used to detect the cell proliferation inhibitory effect of apigenin on MDA-MB-231 cell. ELISA was used to determine the protein level of VEGF secreted by MDA-MB-231 cells. RT-PCR was used to detect mRNA levels of VEGF in MDA-MB-231 cells. The protein levels of HIF-1α, p-AKT, p-ERK1/2, and p53 were detected by Western Blotting. Results： Apigenin did not inhibit the cell viability of MDA-MB-231 cell. Apigenin reduced the secretion and mRNA levels of VEGF in MDA-MB-231 cells. Additionally, apigenin decreased the expressions of HIF-1α, p-AKT and p-ERK1/2, but induced the expression of p53. Conclusion： Apigenin can inhibit VEGF expression in human breast cancer cells, and this may be achieved through decreasing HIF-1α.     

Parathyroid hormone-related protein and human papillomavirus in gynecological tumors

The presence of parathyroid hormone-related protein (PTHrP) and human papillomavirus (HPV) in a series of gynecological tumors from 131 unselected patients was examined. PTHrP was localized immunohistochemically using a highly specific rabbit polyclonal anti-serum against PTHrP(1–16). The results confirmed that gynecological malignancies, although rarely associated with humoral hypercalcemia of malignancy (HHM), stained for PTHrP in a majority of the squamous-cell carcinomas (SCC) at all sites, but only in a minority of adenocarcinomas, and then in areas of squamous metaplasia. This included a series of endometrial tumors. Detection of HPV types was achieved using a polymerase-chain-reaction (PCR) detection system enabling the detection of HPV types 6, 11, 16, 18,31,33 and 45. PTHrP production was not directly related to HPV infection, but correlated with the type of tumor.     

Parathyroid hormone-related protein in breast cancer tissues: Relationship between primary and metastatic sites

The expression of parathyroid hormone-related protein (PTHrP) at primary and metastatic sites was studied retrospectively in specimens obtained at operation and autopsy from 11 patients. The anti-human PTHrP monoclonal antibody, 4B3, was used in the immunohistochemical studies. The 11 cases showed metastases to the liver and the lung, and 9 showed bone metastases at autopsy. At primary sites, PTHrP was positive in the 9 cases with bone metastases, while the other 2 cases were negative for PTHrP. Regardless of the intensities of immunohistochemical staining of PTHrP at primary sites, cancer cells at metastatic sites in the liver and the lung were almost all negative for PTHrP. On the other hand, all PTHrP-positive cases at primary sites at operation showed skeletal metastases at autopsy, and the intensity of the immunohistochemical staining of PTHrP was strongly positive at all the sites of skeletal metastasis. These results suggest that while PTHrP is an important factor that causes cancer cells to erode and grow in skeletal bone, the expression of PTHrP is, concurrently, affected by an osseous microenvironment. Supported in part by Grant-in-Aid from Ministry of Education of Japan.     

Parathyroid hormone-related protein localization in breast cancers predict improved prognosis

In a prospective study of 526 consecutive patients with operable breast cancer, the significance of positive parathyroid hormone-related protein (PTHrP) staining by immunohistology has been evaluated for a median of 10-year follow-up. Improved survival was observed for the 79% of tumors which stained positively for PTHrP [estimated univariate hazard ratio, 0.43; 95% confidence interval (95% CI), 0.30-0.62; P < 0.001]. Adjustments for N stage, progesterone receptor status, and log tumor size changed this estimate only slightly to 0.47 (95% CI, 0.63-0.69; P = 0.001). Patients with PTHrP-positive primary tumors were less likely to develop bone metastases (hazard ratio, 0.63; 95% CI, 0.41-0.98; P = 0.04). PTHrP status was associated with estrogen receptor (P = 0.01), progesterone receptor (P = 0.03), and menopausal status (P = 0.006) but was not significantly associated with tumor size, vascular invasion, tumor grade, or patient age. Of 19 patients requiring surgery for bone metastases, the primary cancers were PTHrP negative in seven, all but one of whom had PTHrP-positive bone metastases. All 12 patients with PTHrP-positive primary cancers also had positive bone metastases. We conclude that increased production of PTHrP by breast cancers confers on them a less invasive phenotype, an effect distinct from the bone resorption-stimulating action that favors bone metastasis. It is likely that the latter property is influenced by factors in the bone microenvironment.     

Immunohistochemical localization of parathyroid hormone-related protein in human breast cancer

Parathyroid hormone-related protein (PTHrP) is known to be a causative factor in humoral hypercalcemia of malignancy. A polyclonal rabbit antiserum directed against the amino-terminal region of the protein and immunoperoxidase methods have been used to locate the presence of PTHrP in a series of 102 consecutive invasive breast tumors removed surgically from normocalcemic women. Positive PTHrP staining was detected in 60% of the tumors but not in the accompanying normal breast tissue. Positive staining was related to the progesterone receptor status of the tumor (P = 0.039) and to the prognostic index of the patient (P = 0.046) and not to estrogen receptor status, patient age, tumor size, histological grade, or nodal status.     

细胞周期蛋白E小干扰RNA靶向调控乳腺癌细胞的生长

目的 研究乳腺癌细胞中细胞周期蛋白E(cyelin E)对乳腺癌细胞肿瘤生物学特征的影响.方法 用带有cyclin E小干扰RNA(siRNA)的真核表达载体pEGFP/CCNE2转染乳腺痛细胞系MCF-7.采用逆转录聚合酶链反应(RT-PCR)和Western blot方法分别在RNA水平和蛋白水平检测siRNA对细胞内cyclin E表达水平的影响,CCK-8方法检测转染后细胞株生长增殖能力以及对化疗药物的敏感性,流式细胞仪检测细胞周期的变化,裸鼠移植瘤成瘤实验检测转染后细胞成瘤能力的变化.结果 pEGFP/CCNE2转染后,MCF-7细胞内cyclin E的表达受到抑制,cyclin E mRNA相对表达水平为0.23±0.05,蛋白相对表达水平为0.24±0.05;细胞的生长增殖能力下降,抑制率为68.56%±0.08%,对化疗药物的敏感性增加;细胞被大量阻滞在G1期,G1期细胞占77.38%.细胞的成瘤能力降低,移植瘤体积缩小.结论 抑制乳腺癌细胞中cyclin E的表达能够抑制乳腺癌细胞的生长、分化和增殖,提高肿瘤对化疗药物的敏感性.     

DNA and oestradiol receptor distribution in human breast cancer.

The content of DNA and oestradiol receptors was measured in two different samples taken from the same tumoral mass of 33 primary infiltrating ductal carcinomas. The results were related to the total cytoplasmic protein and to the DNA of the tissue. The results showed that variations of both DNA and oestradiol receptors are frequent and often significant. The reliability of analyses carried out on a single biopsy is discussed.     

The parathyroid hormone-related protein receptor is expressed in breast cancer bone metastases and promotes autocrine proliferation in breast carcinoma cells

Overproduction of parathyroid hormone-related protein (PTHRP) occurs in a high proportion of primary breast cancers (PBC) and is strongly implicated in their metastatic spread to bone. Although the PTHRP-receptor (PTHRP-R) is often coexpressed with PTHRP in PBC, its role in regulating breast cancer cell proliferation and metastases to bone remains unclear. The aims of this study were to determine the expression of the PTHRP-R in breast cancer bone metastases (BM) and to investigate the effects of PTHRP-R overexpression on breast cancer cell proliferation. PTHRP-R expression occurred in 85% (11 out of 13) of BM compared with 58% (39 out of 67) of PBC. Median expression was higher (P<0.05) in BM compared with PBC. PTHRP increased cAMP accumulation and DNA synthesis in MCF-7 cells stably overexpressing the PTHRP-R (MCF-7(WTR)) but not in MCF-7(VEC) control cells. The increase in DNA synthesis was mimicked by the cAMP pathway activator forskolin. The receptor antagonist PTHRP(7-34) reduced DNA synthesis in MCF-7(WTR) cells, but not MCF-7(VEC) cells, indicating that receptor overexpression promotes autocrine PTHRP activity. MCF-7(WTR) cells showed increased mitogenic responsiveness to fetal calf serum and reduced doubling times. PTHRP induced weak activation of ERK1 and ERK2 and potentiated their activation by serum growth factors. Collectively these results show that the PTHRP-R is frequently expressed in breast cancer BM and indicate that receptor overexpression drives proliferation via autocrine signals that are mediated via cAMP and ERK pathways.     

Parathyroid hormone-related protein regulates the growth of orthotopic human lung tumors in athymic mice.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has growth regulatory effects for many malignant cells and may influence the progression of carcinomas of the breast, prostate, and lung. In the current study, the authors investigated the in vivo and in vitro effects of PTHrP neutralizing antibody and PTHrP treatment on the growth of BEN cells, a human lung squamous cell carcinoma line that expresses PTHrP and its receptor. METHODS: Orthotopic lung tumors were produced in 20 athymic mice with BEN-GFP cells (a clonal line that stably expresses green fluorescent protein [GFP]) by instilling suspensions of 3 x 10(6) cells per mouse into the lungs of anesthetized animals. The mice were divided into 2 groups receiving either subcutaneous mouse antihuman PTHrP antibodies or irrelevant mouse immunoglobulin (Ig) G (150 microg) twice weekly. RESULTS: After 30 days, 6 of 10 mice receiving anti-PTHrP antibodies had lung tumors visible on macroscopic inspection, but only 1 of the 10 mice treated with irrelevant IgG had a lung tumor that was of that size (P < 0.01). GFP fluorescence was significantly greater in lung homogenates of the PTHrP antibody-treated mice than in the mice treated with IgG (6006 +/- 411 vs. 2907 +/- 282 relative fluorescent units, respectively; P < 0.001). Although neutralizing antibodies stimulated BEN cell lung tumor growth, exogenous PTHrP 1-34 treatment (0.01-1 nM) inhibited the growth of cultured BEN cells by approximately 40%. CONCLUSIONS: Although PTHrP expression has been reported to be associated with more aggressive malignancies, the data from the current study suggest that PTHrP 1-34 was a paracrine growth inhibitor in BEN human lung carcinoma cells. The growth-related effects of PTHrP are complex, and can be both stimulatory and inhibitory.     

Sulforaphane inhibits the growth of KPL-1 human breast cancer cells in vitro and suppresses the growth and metastasis of orthotopically transplanted KPL-1 cells in female athymic mice

The anticancer effects of sulforaphane (SFN), which is found in cruciferous vegetables, were studied on KPL-1 human breast cancer cells in vitro and in vivo. Cell proliferation in vitro was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and tumor growth and metastasis in vivo were examined in orthotopically (right thoracic mammary fat pad) transplanted KPL-1 cells in female athymic BALB/c mice. The MTT assay showed that SFN directly inhibited KPL-1 cell growth in vitro (IC50 at 48 h, 19.1 μM; IC50 at 72 h, 17.8 μM). Athymic mice received a KPL-1 cell transplant, and SFN treatment (intraperitoneal injection of 25 or 50 mg/kg SFN) was started the next day. Mice received five injections each week during the 26-day experimental period (for a total of 20 injections). Compared with the SFN-untreated controls, SFN suppressed primary tumor growth. At the termination of the experiment, the final tumor volume was 686±94 mm3 for the control group, 516±70 mm3 (75% of control value) for the 25 mg/kg SFN group and 351±55 mm3 (51% of control value) for the 50?mg/kg SFN group. The final tumor weight was 571±69 mg in the control group, 416±63 mg (73% of the control value) in the 25 mg/kg SFN group and 338±56 mg (59% of the control value) in the 50 mg/kg SFN group. SFN caused a dose-dependent decrease in the proliferation ratio and an increase in the apoptotic ratio of the primary tumor cells. SFN treatment tended to reduce regional (axillary) lymph node metastasis. Treatment with 50 mg/kg SFN significantly inhibited KPL-1 cell growth in vivo by suppressing cell proliferation and inducing apoptosis, and it tended to reduce axillary lymph node metastasis of KPL-1 human breast cancer cell xenografts in female athymic mice.     

Invasion of metastatic human follicular thyroid cancer is inhibited via antagonism of protein kinase C

Signal transduction of a human follicular thyroid cancer cell line (FTC133) was investigated. The protein kinase C (PKC)-agonist TPA enhanced invasion by 15%, whereas its antagonists staurosporine, chelerythrine and calphostin C were inhibiting by up to 62%. TSH and EGF stimulated invasion of FTC133. Antagonism of PKC reversed TSH-mediated stimulation, whereas it had no effect on EGF-stimulation. Our data provide evidence for an essential role of PKC in signal transduction of invasive thyroid cancer.     

Parathyroid hormone-related protein varies with sex and androgen status in nonsmall cell lung cancer

BACKGROUND: In nonsmall cell lung cancer, tumor parathyroid hormone-related protein (PTHrP) expression predicts longer survival in women but not in men. To explain the sex-dependent survival effect, the authors proposed that hormonal influences decrease PTHrP in men versus women, that PTHrP inhibits tumor growth, and that the effect is greater in women than in men. The objectives of this study were to compare lung carcinoma PTHrP expression and carcinoma growth in male and female mice and to determine whether gonadal steroids regulate PTHrP in lung cancer cells. METHODS: Tumor PTHrP content was measured by immunoassay, and tumor burden was assessed with multiple measures in BEN squamous cell orthotopic lung carcinomas in athymic mice. In addition, lung adenocarcinoma PTHrP messenger RNA (mRNA) values determined by microarray analyses were compared between men and women. Cultured lung cancer cells were assayed for PTHrP after treatment with estradiol or R1881, a synthetic androgen. RESULTS: Lung carcinomas contained approximately 3 times more PTHrP in female mice than in male mice. Similarly, levels of PTHrP mRNA were significantly greater in adenocarcinomas from patients who were women than from patients who were men. Male mice had greater tumor burden than female mice. Androgen treatment reduced PTHrP in 3 lung cancer lines. Estradiol had no effect. Testosterone treatment also reduced lung carcinoma PTHrP in female mice. CONCLUSIONS: Lung carcinomas in females expressed more PTHrP than in males possibly because of negative regulation by androgens in males. Female mice with higher tumor PTHrP content had significantly less tumor burden than male mice, supporting the hypothesis that PTHrP inhibits tumor growth.     

Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70

The Myc oncoprotein serves a dual function by stimulating cells both towards growth and apoptosis. The latter functions are often abrogated during tumor development. The Hsp70 stress protein is a potent anti-apoptotic molecule, but its potential role in protecting cells from Myc-mediated apoptosis has not been investigated. Our results show that activated Myc potentiated apoptosis induced by the cancer drugs etoposide (ETO) and camptothecin (CAMP) in v-Myc-expressing human U-937 monoblastic cells and in Rat1 cells containing a conditionally active Myc/estrogen receptor (MycER) fusion protein. However, both heat shock and ectopic Hsp70 expression protected the cells from Myc-mediated apoptosis after drug treatment in both systems. The increased susceptibility to the anti-tumor drugs by activated Myc was enhanced by siRNA-mediated knockdown of Hsp70 expression in U-937 cells. Addressing the mechanisms by which Myc and Hsp70 promotes and inhibits drug-induced apoptosis, respectively, we found that v-Myc stimulated cytochrome c release and activation of effector caspase-9, -3 and -7, but not of initiator caspase-8. Inhibition of caspase-9 specifically reduced v-Myc-stimulated apoptosis, whereas inhibition of caspase-8 and -3/7 reduced apoptosis both in v-myc-expressing and parental ETO-treated U-937 cells. Interestingly, Myc-stimulated activation of effector caspases was inhibited, but cytochrome c release was not affected by Hsp70 expression, suggesting that Hsp70 interferes with the proapoptotic function of Myc downstream of mitochondria, at the level of caspase-9 and downstream caspases. In conclusion, Hsp70 seems to have key function in inhibition of apoptosis mediated by Myc and may therefore play an important role in Myc-driven oncogenesis.