Luteolin suppresses androgen receptor-positive triple-negative breast cancer cell proliferation and metastasis by epigenetic regulation of MMP9 expression via the AKT/mTOR signaling pathway.

BACKGROUND: Triple-negative breast cancer (TNBC) represents up to 20% of all breast cancers. This cancer lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The current therapeutic strategy for patients with this subtype is the use of cytotoxic chemotherapy and surgery. Luteolin is a natural herbal flavonoid and a potential therapeutic candidate for multiple diseases. The use of a treatment that combines Chinese herbal medicine and western medicine is rising in Asia. PURPOSE: The present study evaluates the effects and molecular mechanisms involved with luteolin treatment and evaluates whether this herb affects androgen receptor-positive breast cancer cell proliferation or metastasis. STUDY DESIGN: In vitro evaluation of the effect of luteolin on androgen receptor-positive TNBC cell proliferation and metastasis METHODS: Cell viability analysis was used for the cytotoxicity test. Colony formation and Bromodeoxyuridine (BrdU) staining-based proliferation experiments were used for cell proliferation. Wound healing and transwell assays were used for in vitro migration/invasion. The RT-qPCR analysis was used for gene expression. Furthermore, ChIP-qPCR analysis was used for epigenetic modification of gene promoters. RESULTS: Luteolin significantly inhibited the proliferation and metastasis of androgen receptor-positive TNBC. Furthermore, luteolin inactivated the AKT/mTOR signaling pathway and reversed the epithelial-mesenchymal transition (EMT). The combination of luteolin and inhibitors of AKT/mTOR synergistically repressed an androgen receptor-positive TNBC cell proliferation and metastasis. Luteolin also downregulated MMP9 expression by decreasing the levels of the AKT/mTOR promoting H3K27Ac and H3K56A on the MMP9 promoter region. CONCLUSION: Our findings indicate that luteolin inhibited the proliferation and metastasis of androgen receptor-positive TNBC by regulating MMP9 expression through a reduction in the levels of AKT/mTOR-inducing H3K27Ac and H3K56Ac.

MLL3 Induced by Luteolin Causes Apoptosis in Tamoxifen-Resistant Breast Cancer Cells through H3K4 Monomethylation and Suppression of the PI3K/AKT/mTOR Pathway.

Tamoxifen is one of the most common hormone therapy drug for estrogen receptor (ER)-positive breast cancer. Tumor cells with drug resistance often cause recurrence and metastasis in cancer patients. Luteolin is a natural compound found from various types of vegetables and exhibit anticancer activity in different cancers. This study demonstrated that luteolin inhibits the proliferation and induces apoptosis of tamoxifen-resistant ER-positive breast cancer cells. Luteolin also causes cell cycle arrest at the G2/M phase and decreases mitochondrial membrane potential. Besides, luteolin reduces the levels of activated PI3K/AKT/mTOR signaling pathway. The combination treatment of luteolin and PI3K, AKT, or mTOR inhibitors synergistically increases apoptosis in tamoxifen-resistant ER-positive breast cancer cells. Ras gene family (K-Ras, H-Ras, and N-Ras), an activator of PI3K, was transcriptionally repressed by luteolin via induction of tumor suppressor mixed-lineage leukemia 3 (MLL3) expression. MLL3 increases the level of monomethylation of Histone 3 Lysine 4 on the enhancer and promoter region of Ras genes, thus causes repression of Ras expressions. Our finding implies that luteolin was a promising natural agent against tamoxifen resistance of breast cancer.

Aloe-Emodin Enhances Tamoxifen Cytotoxicity by Suppressing Ras/ERK and PI3K/mTOR in Breast Cancer Cells.

Aloe-emodin (AE) is derived from Aloe vera and rhubarb (Rheum palmatum) and exhibits anticancer activities via multiple regulatory mechanisms in various cancers. AE can also enhance the anticancer efficacy of cisplatin, doxorubicin, docetaxel, and 5-fluorouracil; however, its effects remain poorly characterized. MCF-7, MDA-MB-231, MDA-MB-468, BT-474, and HCC-1954 breast cancer cell lines were treated with the indicated conditions of AE, and cell viability assays were performed. The expression levels of signaling proteins were determined by western blot analysis, intracellular reactive oxygen species (ROS), cell cycle distributions, and rates of apoptosis as estimated by flow cytometry. In comparison with other cells, MCF-7 cells were more sensitive to AE treatment; AE enhanced the cytotoxicity of 9[Formula: see text][Formula: see text]g/ml tamoxifen by reducing EGFR, ER[Formula: see text], Ras, ERK, c-Myc, and mTOR protein expression and blocking PI3K and mTOR activation. Finally, although co-treatment of AE with tamoxifen increased intracellular ROS, there were no effects on cell cycle progression. Besides facilitating tamoxifen-induced cell death, AE also enhanced the antiproliferative activity of tamoxifen by blocking Ras/ERK and PI3K/mTOR pathways in breast cancer cells, thus demonstrating the chemosensitizing potential of AE.

Sann-Joong-Kuey-Jian-Tang decreases the protein expression of Mcl­1 and TCTP and increases that of TNF-α and Bax in BxPC­3 pancreatic carcinoma cells.

Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicinal prescription, has been used for the treatment of lymphadenopathy and solid tumors, and has shown therapeutic potential in several human malignant tumor cell lines. However, the efficacy and molecular mechanisms of action of SJKJT in human pancreatic cancer have not yet been elucidated. In the present study, we evaluated the cytotoxic effects of SJKJT on BxPC-3 human pancreatic carcinoma cells by MTT assay. The protein expression levels of myeloid cell leukemia 1 protein (Mcl-1), translationally controlled tumor protein (TCTP), tumor necrosis factor-α (TNF­α), caspase-8, caspase-3, Bax and Bcl-2 family in the BxPC-3 cells were measured by western blot analysis. The cell cycle was analyzed by flow cytometry. The protein expression of caspase-3 was also detected by immunocytochemistry (ICC). The results revealed that SJKJT inhibited the proliferation of BxPC-3 cells in a time- and dose-dependent manner. The protein expression levels of TNF-α, caspase-8, caspase-3 and Bax increased in the BxPC-3 cells treated with SJKJT; however, the levels of Mcl-1, TCTP and Bcl-xL decreased. The results also demonstrated that SJKJT increased the percentage of BxPC-3 cells in the sub-G1 phase. In addition, ICC staining indicated that the protein expression of caspase-3 was upregulated in the BxPC-3 cells treated with SJKJT. These findings indicate that SJKJT inhibits the proliferation of BxPC-3 cells through the extrinsic and intrinsic pathway, inducing apoptosis in vitro. Our study, using BxPC-3 human pancreatic cancer cells, demonstrates that SJKJT has potential as a chemotherapeutic agent for the treatment of pancreatic cancer. Further sutdies are warranted to fully elucidate its mechanisms of action.

Sann-Joong-Kuey-Jian-Tang inhibits hepatocellular carcinoma Hep-G2 cell proliferation by increasing TNF-α, Caspase-8, Caspase- 3 and Bax but by decreasing TCTP and Mcl-1 expression in vitro.

Hepatic cancer remains a challenging disease and there is a need to identify new treatments. Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional medicinal prescription, has been used to treat lymphadenopathy and exhibits cytotoxic activity in many types of human cancer cells. Our previous studies revealed that SJKJT is capable of inhibiting colon cancer colo 205 cells by inducing autophagy and apoptosis. However, the effects and molecular mechanisms of SJKJT in human hepatocellular carcinoma have not been clearly elucidated. In the present study we evaluated the effects of SJKJT in human hepatic cellular carcinoma Hep-G2 cells. The cytotoxicity of SJKJT in Hep-G2 cells was measured by MTT assay. The cell cycles were analyzed by fluorescence­activated cell sorting (FACS). The protein expression of translationally controlled tumor protein (TCTP), Mcl-1, Fas, TNF-α, Caspase-8, Caspase-3 and Bax in Hep-G2 cells treated with SJKJT was evaluated by western blotting. The protein expression of Caspase-3 was also detected by immunofluorescence staining. The results showed that SJKJT inhibits Hep-G2 cells in a time- and dose­dependent manner. During SJKJT treatment for 48 and 72 h, the half-maximum inhibitory concentration (IC50) was 1.48 and 0.94 mg/ml, respectively. The FACS results revealed that increased doses of SJKJT were capable of increasing the percentage of cells in the sub-G1 phase. Immunofluorescence staining showed that Hep-G2 treated with SJKJT had increased expression of Caspase-3. The western blot results showed that the protein expression of Fas, TNF-α, Caspase-8, Caspase- 3 and Bax was upregulated, but that of TCTP and Mcl-1 was downregulated in Hep-G2 cells treated with SJKJT. In conclusion, these findings indicated that SJKJT inhibits Hep-G2 cells. One of the molecular mechanisms responsible for this may be the increased Fas, TNF-α, Caspase-8, Caspase- 3 and Bax expression; another mechanism may be via decreasing TCTP and Mcl-1 expression in order to induce apoptosis.

Anticancer effects of eleven triterpenoids derived from Antrodia camphorata.

Eleven derivatives from Antrodia camphorata were isolated in order to evaluate their selective cytotoxicity toward 14 types of human cancer cell and two non-transformed cell types. Among these triterpenoids, methyl antcinate A (MAA) exhibited the most potent spectrum of anticancer effects in KB cells, four different oral cancer cell lines (TSCCa, GNM, OC-2, and OEC-M1), Panc-1, BT474, PC-3, OVCAR-3, HeLa, and U2OS cells with high selectivity indices (CC(50)/IC(50)). The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and poly(ADP-ribose) polymerase (PARP) of PC-3 cells tested by western blotting suggested that MAA exerts cell death through the caspase-dependent cascade and the Bax-mediated mitochondrial apoptotic pathway, not only on liver and oral cancer cells but on other types as well, including prostate cancer, in a dose-dependent manner. In addition to MAA, methyl antcinate B, dehydroeburicoic acid, and 15α-acetyl-dehydrosulfurenic acid also exhibited significant selective cytotoxic effects to respective cancer cells. Modifications of these triterpenoids may lead to the development of more potent anticancer drugs.

The Potential Utility of Curcumin in the Treatment of HER-2-Overexpressed Breast Cancer: An In Vitro and In Vivo Comparison Study with Herceptin.

HER-2 is an important oncoprotein overexpressed in about 15-25% of breast cancers. We hypothesized that the ability of curcumin to downregulate HER-2 oncoprotein and inhibit the signal transduction pathway of PI3K/Akt, MAPK, and NF-κB activation may be important in the treatment of HER-2-overexpressed breast cancer. To examine the effect of curcumin on breast cancer cells, MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr (a herceptin resistant strain from SK-BR-3) cells were used for in vitro analysis. The in vivo effect of curcumin on HER-2-overexpressed breast cancer was investigated with the HER-2-overexpressed BT-474 xenograft model. Cell growth, cell cycle change, the antimobility effect, signal transduction, and xenograft volume analysis between groups treated with herceptin and/or curcumin were tested. Curcumin decreased the cell growth of various breast cancer cell lines (MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr). In Western blot analysis, the phosphorylation of Akt, MAPK, and expression of NF-κB were reduced in BT-474 cells, but not in SK-BR-3-hr cells, after treatment with herceptin. When treated with curcumin, the HER-2 oncoprotein, phosphorylation of Akt, MAPK and expression of NF-κB were decreased in both BT-474 and SK-BR-3-hr cells. In the BT-474 xenograft model, though not as much as herceptin, curcumin did effectively decrease the tumor size. The combination of curcumin with herceptin was not better than herceptin alone; however, the combination of taxol and curcumin had an antitumor effect comparable with taxol and herceptin. The results suggested that curcumin has potential as a treatment for HER-2-overexpressed breast cancer.

Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study.

Tanshinone IIA (Tan IIA) and sodium tanshinone IIA sulfonate (STS) were found to have protective effects on cardiomyocyte against adriamycin-induced damage and may be used clinically. It is unclear whether the supplementation of STS or Tan IIA would affect the anticancer activity of anthracycline. To evaluate the effect of Tan IIA or STS on the anticancer of epirubicin, the cell viability, apoptosis, Akt expression, and uptake of epirubicin after supplementation of Tan IIA or STS in the epirubicin-treated BT-20 cells were measured and compared. Tan IIA inhibited BT-20 cell growth and induced apoptosis in a time- and dose-dependent manner. When Tan IIA was used with epirubicin, an increase of BT-20 cells apoptosis was accompanied by the decreasing phosphorylation of Akt. STS had no effect on the cell viability of BT-20 cells. However, when used with epirubicin, STS decreased the epirubicin-induced cytotoxicity and apoptosis in BT-20 cells. The antagonistic effect of STS on epirubicin-induced cytotoxicity in BT-20 cells occurred concomitantly with the reduced epirubicin uptake and the increased phosphorylation of Akt. STS decreased the uptake of epirubicin in BT-20 cells and blocked epirubicin-induced apoptosis through activation of Akt.

Acupuncture-related rapid dermal spread of breast cancer: a rare case.

Many ethnic Chinese patients seek second or adjuvant alternative therapies after breast cancer is diagnosed. Chinese herbs and acupuncture are the most popular methods in East Asia. We present a case of acupuncture manipulation-related cutaneous spread that no literature reported before. Post-acupuncture cutaneous spread was noted in a 54-year-old woman with left neck lymph node recurrence after complete surgery, chemotherapy and radiotherapy treatment. The results of chest computed tomography and skin biopsy showed the metastatic breast cancer in the dermis. Six courses of paclitaxel and gemcitabine followed by trastuzumab were given as therapeutic chemotherapy. The neck mass and cutaneous lesions subsided after 2 courses of chemotherapy. Direct puncture of a metastatic lymph node might increase the incidence of tumor spread on the skin. Therefore, despite the efficacy of complementary and alternative medicine, its safety and possible side effects should be more emphasized.

Serum and tissue trace elements in patients with breast cancer in Taiwan.

The objective of this study was to compare levels of four elements (zinc, copper, selenium, and iron) in the serum and tissue of 68 breast tumor patients (benign and malignant), from a teaching hospital in central Taiwan. Samples of normal tissue (5 cm away from tumor) were also taken from patients with malignant tumors. Only serum was taken from the 25 healthy persons in the control group. Results showed that Zn, Cu, Se, Fe, Cu/Zn, Cu/Se, and Cu/Fe were present in different amounts in the serum of each of the three groups. Zn and Se levels were lower in the serum of the two tumor groups compared to the control group. In tissue samples, Zn, Cu, Se, and Fe concentrations were different in each of the three groups. The malignant tissue had the highest levels of all four elements. In advanced-stage malignant tumors, levels of Cu and the ratios of Cu/Fe and Cu/Zn (in both serum and tissue) were highest. The ratios of serum Cu/Zn, Cu/Fe, and Cu/Se were also higher in malignant patients. The cutoff value of serum Cu/Zn was 1.2 (sensitivity and specificity were both 100%). The Cu/Zn ratio was highest in the advanced stages of cancer and was a better diagnostic tool for breast cancer than Cu/Se and Cu/Fe. The authors suggest that change of trace elements in serum and tissue might be useful and significant as biomarkers involving the initial plastic process.