ABSTRACT
The herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) are widely used in Chinese medicine to treat several aliments and as an adjuvant to chemotherapy of lung cancer. No information is available regarding the two herbs' influence on ovarian and endometrial cancer. To fill this data gap we compared cell growth responses to SB and FC in ovarian and endometrial cancer cell lines. Dose-dependent cell growth inhibition was observed following higher doses in all cell lines while lower doses stimulated growth in only endometrial cell lines. Higher doses of SB and FC significantly decreased cell growth on soft agar and decreased the invasive potential of cancer cells. Treatment of cells with both herbs resulted in activation of caspase-3, G0 /G1 phase cell cycle arrest, downregulation of cyclins D1 and D3 and induction of p27. Both herbs decreased NFκB DNA binding, reduced expression of phosphorylated IκBα, abrogated NFκB activation, and downregulated NFκB-regulated metastasis-promoting proteins in cancer cells. Furthermore, knockdown of NFκB attenuated SB- and FC-induced cell growth inhibition. These results suggest that inhibition of NFκB activation may be an important mechanism for growth suppression by SB and FC. Data indicate that these herbs may represent a new source of agents for NFκB inhibition in cancer therapy.
Subject(s)
Cell Proliferation/drug effects , Drugs, Chinese Herbal/pharmacology , Endometrial Neoplasms/pathology , Fritillaria/chemistry , NF-kappa B/antagonists & inhibitors , Ovarian Neoplasms/pathology , Phytotherapy , Scutellaria baicalensis/chemistry , Apoptosis/drug effects , Blotting, Western , Cell Cycle/drug effects , Cell Movement/drug effects , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/metabolism , Female , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , RNA, Small Interfering/genetics , Tumor Cells, CulturedABSTRACT
Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol, and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. Combination treatment with both agents enhanced vitamin D receptor expression and inhibited cell proliferation through caspase-3 activation and induction of G0-G1 cell-cycle arrest with associated downregulation of cyclins D1 and D3 and p27 induction. We used mass spectrometry-based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression among these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2-associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone, or the combination. Further BAX analysis through gain- or loss-of-function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:BCL-2 ratio. Knockdown of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol upregulate the expression of BAX along with other apoptosis-related proteins, which induce inhibition of endometrial cancer cell growth by apoptosis and cell-cycle arrest.
Subject(s)
Apoptosis/drug effects , Biomarkers, Tumor/metabolism , Calcitriol/pharmacology , Endometrial Neoplasms/pathology , Progesterone/pharmacology , Receptors, Calcitriol/metabolism , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Chromatography, Liquid , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/metabolism , Endometrium/cytology , Endometrium/drug effects , Endometrium/metabolism , Female , Histones/metabolism , Humans , Mitochondrial Proteins/metabolism , Proteomics , Tandem Mass Spectrometry , bcl-2-Associated X Protein/metabolism , eIF-2 Kinase/metabolismABSTRACT
Cytokines/chemokines are key players in cancer-related inflammation. Increasing evidence suggests that chemokines produced by tumor cells are the mediators of metastasis. Thus, agents that can downregulate chemokines expression have potential against cancer metastasis. We have previously shown inhibition of ovarian and endometrial cancer cell growth with progesterone and calcitriol. In the present study, we evaluated the effect of these two agents on the expression of inflammatory genes. Using a RT-PCR array of inflammatory cytokines/chemokines and their receptors, we found a marked attenuation of CXCL1 and CXCL2 (GRO-α and -ß) in cancer cells by both treatments. Knockdown of NFκB resulted in a reduced expression of CXCL1 and CXCL2 and the inhibitory effect of progesterone and calcitriol on the expression of chemokines was abrogated in NFκB-silenced cancer cells. Silencing of IκBα increased the expression of CXCL1 and CXCL2 in cancer cells, which can be attributed to the increased activation of NFκB-p65, caused by the lack of its inhibitor. Progesterone and calcitriol-induced inhibition was abolished in IκBα-knockdown cells. Our results demonstrate that suppression of IκBα phosphorylation by progesterone and calcitriol contributes to the reduced expression of CXCL1 and CXCL2. Downregulation of CXCL1 and CXCL2 was associated with a marked inhibition of metastasis-promoting genes. Overall, our results indicate that progesterone and calcitriol inhibit IκBα phosphorylation, NFκB activation, and the expression of NFκB regulated metastasis promoting genes. These results provide attractive data for the possible use of progesterone and calcitriol in the management of endometrial and ovarian tumors.
Subject(s)
Calcitriol/pharmacology , Chemokine CXCL1/metabolism , Chemokine CXCL2/metabolism , Endometrial Neoplasms/pathology , Ovarian Neoplasms/pathology , Progesterone/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Chemokine CXCL1/genetics , Chemokine CXCL2/genetics , Drug Screening Assays, Antitumor , Endometrial Neoplasms/genetics , Endometrial Neoplasms/metabolism , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Gene Silencing , Humans , I-kappa B Proteins/genetics , I-kappa B Proteins/metabolism , NF-KappaB Inhibitor alpha , Neoplasm Metastasis/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Phosphorylation/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , TransfectionABSTRACT
Class 3 semaphorins (SEMA), SEMA3B and SEMA3F, are secreted proteins that regulate angiogenesis, tumor growth, and metastasis by binding to their transmembrane receptor complex consisting of plexins and neuropilins (NP). Expression of SEMAs and their receptors was assessed in tissue microarrays by immunohistochemistry. SEMA3B, SEMA3F, and plexin A3 were expressed strongly in normal endometrial tissues, whereas grade-dependent decreases were found in endometrial carcinomas. No change was observed in the expression of plexin A1, NP1, and NP2 in normal versus endometrial cancer tissues. Endometrial cancer cells showed decreased expression of SEMA3B, SEMA3F, and plexin A3 compared with their normal counterparts. Treatment of cancer cells with progesterone (P4) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] for a period of 72 hours induced a significant upregulation of SEMA3B and SEMA3F as well as inhibited growth of cancer cells by increasing caspase-3 activity. Cotreatment of cell lines with P4 or 1,25(OH)(2)D(3) and their respective antagonists confirmed the specificity of their actions. Transfection of siRNA-targeting SEMA3B and SEMA3F in endometrial cancer cells attenuated P4 or 1,25(OH)(2)D(3)-induced growth inhibition. Restoration of SEMA3B or SEMA3F expression in cancer cells caused growth inhibition, reduced soft agar colony formation, and cell invasiveness by inhibiting expression of matrix metalloproteinase-2 (MMP-2), MMP-9, integrin αvß3, and proangiogenic genes and by upregulating antiangiogenic genes. Thus, we have identified two new P4 and 1,25(OH)(2)D(3)-regulated antitumor genes for endometrial cancer. These results suggest that the loss of SEMAs contribute to the malignant phenotype of endometrial cancer cells and that reexpression of SEMAs by ectopic expression or with anticancer agents P4 or 1,25(OH)(2)D(3) can be a promising therapeutic treatment against endometrial cancer.
