Tanshinone IIA can inhibit MiaPaCa­2 human pancreatic cancer cells by dual blockade of the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways.

Tanshinone IIA (Tan­IIA; C19H18O3) is derived from Danshen (the roots of Salvia miltiorrhiza), and has been reported to possess anti­inflammatory and antioxidant activities. Tan­IIA can inhibit BxPC­3 human pancreatic cancer cells in vitro through inducing endoplasmic reticulum stress and apoptosis via mitochondrial pathways. However, the efficacy and molecular mechanisms of Tan­IIA in human pancreatic cancer have not yet been elucidated. The transmembrane tyrosine kinases, including insulin­like growth factor 1 receptor (IGF1R), vascular endothelial growth factor receptor (VEGFR) or epidermal growth factor receptor (EGFR) have been implicated in the survival and metastasis of cancer. In addition, the Ras/Raf/mitogen­activated protein kinase kinase (MEK)/extracellular signal­regulated kinase (ERK) and phosphoinositide 3­kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways are the most commonly dysregulated kinase cascades in human cancer. The present study aimed to investigate the efficacy and molecular mechanisms of Tan­IIA in MiaPaCa­2 human pancreatic carcinoma cells. The protein expression levels of EGFR, IGF1R, VEGFR, Ras, PI3K, AKT, mTOR, Raf, MEK, ERK and phosphatase and tensin homolog (PTEN) were detected in Tan­IIA­treated MiaPaCa­2 cells by western blotting. The results demonstrated that the protein expression levels of EGFR, IGF1R, VEGFR, Ras, Raf, MEK, ERK, PI3K, AKT, mTOR and PTEN were decreased in MiaPaCa­2 cells treated with various concentrations of Tan­IIA for different durations. In conclusion, these findings indicated that Tan­IIA may inhibit MiaPaCa­2 human pancreatic cancer cells; the molecular mechanisms underlying this inhibitory effect may be involved in downregulating EGFR, IGF1R and VEGFR expression, and dual blockade of the Ras/Raf/MERK/ERK and PI3K/AKT/mTOR pathways.

Tanshinone IIA inhibits gastric carcinoma AGS cells by decreasing the protein expression of VEGFR and blocking Ras/Raf/MEK/ERK pathway.

The RAS/RAF/MEK/ERK pathway is one of the most frequently dysregulated kinase cascades in human cancer, that facilitate the proliferation and survival of cancers driven by growth factor receptors. Tanshinone IIA (Tan-IIA) was extracted from Danshen (Salviae Miltiorrhizae Radix). Tan-IIA inhibition of the proliferation of gastric cancer are well documented, but the molecular mechanisms of Tan-IIA inhibition of gastric cancer have not been well elucidated. We evaluated the protein expression of vascular epidermal growth factor receptor (VEGFR), human epidermal growth factor receptor 2 (HER2), Ras, Raf, MEK, ERK, PARP, caspase-3 and ß-actin in AGS cells by western blotting. The results showed that AGS cells treated with Tan-IIA upregulated the protein expression of PARP and caspase-3 but decreased VEGFR, HER2, Ras, Raf, MEK and ERK time- and dose-dependently. These findings demonstrated that Tan-IIA inhibited human gastric cancer AGS cells; one of the molecular mechanisms may be through decreasing the protein expression of VEGFR and HER2, then blocking the Ras/Raf/MEK/ERK pathway to induce the activation of PARP and caspase-3 to induce apoptosis.

Tanshinone IIA increases protein expression levels of PERK, ATF6, IRE1α, CHOP, caspase­3 and caspase­12 in pancreatic cancer BxPC­3 cell­derived xenograft tumors.

Tanshinone (Tan)-IIA is a derivative of phenanthrenequinone and the main active ingredient isolated from Salviae miltiorrhizae radix (Danshen). Previous studies have demonstrated that Tan­IIA increased the protein expressions levels of protein kinase RNA­like endoplasmic reticulum kinase (PERK), activating transcription factor (ATF) 6, caspase­12 and CCAAT­enhancer­binding protein homologous protein (CHOP), to induce endoplasmic reticulum (ER) stress and apoptosis in human pancreatic cancer BxPC­3 cells. However, to the best of our knowledge, the effects of Tan­IIA on pancreatic cancer cells have not been investigated in vivo. Further studies are required to elucidate the therapeutic potential of Tan­IIA in inducing ER stress in cancer cells in vivo. The present study aimed to investigate the effects of Tan­IIA on the expression of ER stress­related proteins in BxPC­3­derived xenograft tumors. A total of 30 male severe combined immunodeficiency mice (age, 4 weeks) were implanted with BxPC­3 cells (2x106/0.2 ml) and subsequently treated with various doses of Tan­IIA (0, 30 and 90 mg/kg) for 4 weeks. After mice were sacrificed on day 33, the xenograft tumors were dissected and total protein was extracted for western blot analysis. The results of the present study demonstrated that Tan­IIA inhibited the growth of BxPC­3­derived xenograft tumors. In addition, Tan­IIA increased the protein expression levels of PERK, ATF6, caspase­12, inositol­requiring enzyme (IRE) 1α, eukaryotic initiation factor (eIF) 2α, phosphorylated (p)­c­Jun N­terminal kinase (JNK), CHOP and caspase­3 in a dose­dependent manner. These results indicated that Tan­IIA induced ER stress via increasing the protein expression levels of PERK, ATF6, caspase­12, IRE1α, eIF2α, p­JNK, CHOP and caspase­3 in BxPC­3 cells in vivo. Therefore, it may be hypothesized that Tan­IIA has potential for the development of novel therapeutic strategies for the treatment of patients with pancreatic cancer.

Effect of Lipodox in combination with bevacizumab in a patient with a metastatic malignant phyllodes breast tumor: A case report.

A 76-year-old female patient with a malignant phyllodes tumor underwent modified radical mastectomy and wide excision. Multiple nodules were observed in the operated wound area. Positron emission tomography-computed tomography (PET-CT) revealed recurrent disease in the left breast, the adjacent left third rib, the left internal mammary region and the left ilium. A novel formulation of bevacizumab (5 mg/m2, first day) in combination with liposomal doxorubicin (Lipodox, 30 mg/m2, second day) was administered for 3 cycles every 2 weeks, and subsequently wide excision was performed. Lipodox (40 mg/m2) was administered for 3 cycles every 3 weeks, starting 4 weeks after the surgery. Follow-up whole body PET-CT scanning, 3 and 6 months later, indicated no sign of residual hypermetabolic malignancy. Malignant phyllodes tumors do not usually respond to chemotherapy or radiotherapy. In the present case report, a novel formulation of bevacizumab in combination with Lipodox was administered as neoadjuvant chemotherapy in a patient with a malignant phyllodes tumor and preoperative tumor shrinkage was achieved, resulting in clear resection margins.

Tanshinone IIA decreases the protein expression of EGFR, and IGFR blocking the PI3K/Akt/mTOR pathway in gastric carcinoma AGS cells both in vitro and in vivo.

Tan-IIA exerts powerful inhibitory effects in gastric cancer AGS cells. The PI3K/AKT/mTOR pathway is one of the most frequently dysregulated kinase cascades in human cancer. In the present study, we investigated the protein expression levels of PI3K, AKT and mTOR in AGS cells treated with Tan-IIA both in vitro and in vivo. The AGS cells were treated with Tan-IIA for different durations in vitro. In the in vivo study, AGS cell xerograft SCID mice were treated with Tan-IIA for 8 weeks. Subsequently, the protein expression of EGFR, IGFR, PI3K, AKT and mTOR was measured by western blotting. The results showed that Tan-IIA was able to decrease the protein expression levels of EGFR, IGFR, PI3K, AKT and mTOR significantly and dose-dependently in vitro and in vivo. In conclusion, these findings indicate Tan-IIA could inhibit AGS cells through decreasing the protein expression of EGFR, IGFR and blocking PI3K/AKT/mTOR pathway both in vitro and in vivo.

Tanshinone IIA decreases the migratory ability of AGS cells by decreasing the protein expression of matrix metalloproteinases, nuclear factor κB-p65 and cyclooxygenase-2.

During progression of gastric cancer, degradation of the extracellular matrix by matrix metalloproteinases (MMPs) has been associated with poor prognosis. Tanshinone IIA (Tan-IIA) exerts antitumor activity in a variety of human cancer cells. It is extracted from Danshen (Salviae miltiorrhizae radix), and induces apoptosis and inhibits the proliferation of gastric cancer cells. However, the molecular mechanisms underlying the inhibition of migration in gastric cancer by Tan-IIA have not been fully elucidated. In the present study, AGS cell migration ability was evaluated using a wound-healing assay. The protein expression levels of nuclear factor (NF)-κB-p65, cyclooxygenase (COX)-2, MMP-2, -7, and -9 and ß-actin in AGS cells were measured by western blotting. The results demonstrated that AGS cells treated with Tan-IIA exhibit decreased protein expression levels of NF-κB-p65, COX-2, and MMP-2, -7 and -9. The results also indicate that Tan-IIA inhibits migration ability in a dose- and time-dependent manner. These findings demonstrate that Tan-IIA inhibits the migration ability of AGS human gastric cancer cells and that decreasing the protein expression of NF-κB-p65, COX-2, and MMP-2, -7 and -9 may be an underlying molecular mechanism.

Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways.

Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer.

Myoid hamartoma of the breast with focal chondromyoxid metaplasia and pseudoangiomatous stromal hyperplasia: A case report.

Hamartomas of the breast, also known as fibroadenolipomas, lipofibroadenomas or adenolipomas, are benign lesions. Hamartomas account for between 0.04 and 1.15% of all benign breast tumors in females. Myoid hamartoma of the breast (MHB) is extremely rare. The present study describes a case of MHB in a 44-year-old female. Screening mammography revealed a lobulated partial indistinct isodense mass measuring ~3.8 cm in the upper outer quadrant of the left breast. Sonographic examinations revealed a 2-3-cm mass in the left breast, which was fairly well circumbscribed and demonstrated complex scattered echogenic areas and isoechoic tissue. A core needle biopsy demonstrated fibrocystic changes, with small focal ductule aggregations. As malignancy could not be excluded, a partial mastectomy was performed using a circumareolar incision. The mass was histopathologically diagnosed as MHB with focal chondromyoxid metaplasia and pseudoangiomatous stromal hyperplasia. The histological diagnosis was based upon the findings of the well-circumscribed tumor, which was composed of entrapped mammary ducts, fat cells and myoid stromal components, with focal chondromyxoid metaplasia and pseudoangiomatous stromal hyperplasia. The tumor cells exhibited diffuse cluster of differentiation 34-positive immunoreactivity, which was consistent with a diagnosis of pseudoangiomatous stromal hyperplasia.

Sann-Joong-Kuey-Jian-Tang decreases the protein expression of mammalian target of rapamycin but increases microtubule associated protein II light chain 3 expression to inhibit human BxPC­3 pancreatic carcinoma cells.

Sann­Joong­Kuey­Jian­Tang (SJKJT), a Traditional Chinese Medicinal prescription, has been used for the treatment of lymphadenopathy and solid tumors, and has shown therapeutic potential in a number of human malignant tumor cell lines, such as Hep­G2 hepatocellular carcinoma cells. Previous mechanistic studies demonstrated that SJKJT inhibited the proliferation of BxPC­3 pancreatic carcinoma cells through the extrinsic and intrinsic apoptotic pathways in vitro. SJKJT was also shown to be cytotoxic to colo 205 colon cancer cells by inducing autophagy in vitro. The present study therefore investigated molecular mechanisms of autophagy in human BxPC­3 pancreatic cancer cells treated with SJKJT. The cytotoxic effects of SJKJT on BxPC­3 human pancreatic carcinoma cells were evaluated using an MTT assay. Furthermore, the expression of autophagy­associated proteins, including mammalian target of rapamycin (mTOR), beclin­1, autophagocytosis­associated protein (Atg)3, Atg7, Atg5­Atg12 and microtubule­associated protein II light chain 3 (LC3­II), was assessed using western blot analysis. The results demonstrated that BxPC­3 cells treated with SJKJT exhibited decreased expression levels of mTOR and increased expression of LC3­II protein. In addition, the expression of the beclin­1, Atg3, Atg7 and Atg5­Atg12 proteins was increased during the first 24 h, but decreased from 48 to 72 h. The results showed that SJKJT inhibited the proliferation of human BxPC­3 pancreatic cancer cells in vitro. A possible underlying molecular mechanism may be the induction of autophagy. Further investigation into the therapeutic potential of SJKJT in human pancreatic cancer is required.

Tanshinone IIA inhibits gastric carcinoma AGS cells through increasing p-p38, p-JNK and p53 but reducing p-ERK, CDC2 and cyclin B1 expression.

Tanshinone IIA (Tan-IIA) is extracted from Danshen (Salviae miltiorrhizae radix). It possesses antitumor activity against a variety of human cancer cells and its induction of apoptosis and inhibition of proliferation of gastric cancer cells are well-documented. However, the molecular mechanisms by which Tan-IIA inhibits gastric cancer have not been well-elucidated. In the present study, we evaluated the cytotoxicity of Tan-IIA against human gastric cancer AGS cells by the (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) MTT assay. The protein expression of tumor necrosis factor-alpha (TNF-α), FAS, p53, p21, cyclin A, cyclin B1, extracellular-related kinase (ERK), phospho extracellular-related kinase (p-ERK), p38, p-p38, Jun-amino-terminal kinase (JNK), phospho Jun-amino-terminal kinase (p-JNK) and ß-actin in AGS cells were measured by western blotting. The cell-cycle distribution was analyzed by flow cytometry. The results showed that Tan-IIA inhibited AGS cells with time- and dose-dependent manners. AGS cells treated with Tan-IIA up-regulated the protein expression of TNFα, FAS, p-p38, p-JNK, p53, p21, caspase-3 and caspase-8 but reduced that of p-ERK, CDC2, cyclin A, and cyclin B1. The results also showed that Tan-IIA dose dependently induced G2/M phase arrest. These findings demonstrate that Tan-IIA can inhibit AGS human gastric cancer cells; one of the molecular mechanisms may be through increasing the protein expression of p-p38 and p-JNK, but decreasing that of p-ERK to induce the activation of p53, followed by increasing the protein expression of p21 to down-regulate CDC2 and cyclin B1 expression which then induces G2/M phase arrest. Another route may be through increasing the protein expression of TNF-α, FAS, caspase-8 and caspase-3 to induce apoptosis.

Tanshinone IIA inhibits human gastric carcinoma AGS cell growth by decreasing BiP, TCTP, Mcl­1 and Bcl­xL and increasing Bax and CHOP protein expression.

Tanshinone IIA (Tan-IIA) is extracted from Danshen (Salviae Miltiorrhizae Radix) and is a natural anti-cancer agent, which possesses antitumor activity in a variety of human cancer cells. Tan-IIA can induce apoptosis and inhibit the proliferation of gastric cancer through different molecular mechanisms. However, the efficacy and molecular mechanism of Tan-IIA in gastric cancer have not been well studied. In the present study, the cytotoxicity of Tan-IIA in human gastric cancer AGS cells by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay was examined. The protein expression levels of B-cell lymphoma-extra large (Bcl-xL), Bcl-2-associated X protein (Bax), myeloid cell leukemia 1 protein (Mcl-1), translationally-controlled tumor protein (TCTP), binding immunoglobulin protein (BiP), calnexin, protein kinase-like endoplasmic reticulum kinase, eIF2α, activating transcription factor 4 (ATF4), inositol-requiring enzyme 1α (IRE1α), ATF6, caspase-12, caspase-9, caspase-3, C/EBP-homologous protein (CHOP) and ß-actin in AGS cells were measured by western blot analysis. The results showed that Tan-IIA inhibited AGS cells in a time-and dose-dependent manner. AGS cells treated with Tan-IIA upregulated the protein expression of caspase-12, caspase-9, caspase--3, CHOP and Bax, but downregulated the protein expression of BiP, TCTP, Mcl-1 and Bcl-xL. These findings indicated that Tan-IIA inhibits the growth of human gastric cancer AGS cells. One of the molecular mechanisms may be through decreasing the protein expression of BiP to induce the activation of endoplasmic reticulum stress, followed by increasing the protein expression of caspase-12 to upregulate CHOP expression. The other may be through decreasing the protein expression of Mcl-1, Bcl-xL and TCTP, but increasing Bax, caspase-9 and caspase-3 to induce apoptosis. The chemotherapeutic potential of Tan-IIA for human gastric cancer warrants further study in the future.

The prognostic significance of metaplastic carcinoma of the breast (MCB)--a case controlled comparison study with infiltrating ductal carcinoma.

PURPOSE: Metaplastic carcinoma of the breast (MCB) is a rare histological subtype of breast cancer with an incidence of less than 0.1%-0.5%. Due to its rarity, the clinical characteristics and prognostic significance of MCB compared with other common breast cancers (like infiltrating ductal carcinoma [IDC], and infiltrating lobular carcinoma [ILC]) are not clear, and controversial among different reports. METHODS: We performed a collective comparison study of multi-institutional cases to evaluate the clinical characteristics and prognostic status of MCB to compare with IDC and ILC. A case control analysis was performed to minimize the bias from clinicopathologic factors between IDC and MCB. Disease free survival (DFS) and overall survival (OS) between groups were compared. RESULTS: Forty-five MCB patients were enrolled from the 4 medical centers and compared with 1777 IDC and 53 ILC patients from the CCH cancer registry database comprise the current study. Compared with IDC, MCB was associated with older age, larger tumor size, a lesser lymph node positive rate, a higher likelihood of distant metastasis, higher tumor grade, lower ER-positive tumor, and higher triple negative breast cancer subtype (TNBC). MCB was associated with worse OS (p = 0.031) than IDC, but no difference in DFS (p = 0.071); however, MCB was not statistically different from ILC in both DFS and OS (p = 0.289 and 0.132, respectively). Compared with the case-controlled IDC group, MCB patients had poorer OS (p = 0.040), but no difference in DFS (p = 0.439). CONCLUSION: MCB is associated with poorer OS than IDC, and this was related to tumor behavior rather than clinicopathologic factors.

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.

Tanshinone IIA inhibits the growth of pancreatic cancer BxPC­3 cells by decreasing protein expression of TCTP, MCL­1 and Bcl­xL.

Pancreatic cancer remains a challenging disease worldwide. Tanshinone IIA (Tan­IIA) is one of the active constituents of Danshen (Radix Salviae miltiorrhizae). Tan­IIA has been hypothesized to inhibit numerous human cancer cells by various molecular mechanisms. However, the efficacy and molecular mechanism of Tan­IIA action in pancreatic cancer has not been well studied. In the present study, the cytotoxicity of Tan­IIA in human pancreatic cancer BxPC­3 cells was evaluated by MTT assay. Cell cycle analysis of BxPC­3 cells treated with Tan­IIA was performed by flow cytometry (FACS). Protein expression levels of TCTP, Mcl­1, Bcl­xL, Bax and Caspase­3 in BxPC­3 cells were measured by western blot analysis. The results revealed that Tan­IIA inhibited BxPC­3 cells in a time­ and dose­dependent manner. FACS analysis demonstrated that Tan­IIA increases the rate of sub­G1 phase. BxPC­3 cells treated with Tan­IIA were identified to upregulate protein expression of Bax and Caspase­3 and downregulate expression of TCTP, Mcl­1 and Bcl­xL. These results indicate that Tan­IIA may inhibit BxPC­3 human pancreatic cancer cells through the induction of apoptosis by decreasing protein expression of TCTP, Mcl­1 and Bcl­xL and increasing Bax expression in vitro. The chemotherapeutic potential of Tan­IIA for human pancreatic cancer warrants further study.

Tanshinone IIA potentiates the efficacy of 5-FU in Colo205 colon cancer cells in vivo through downregulation of P-gp and LC3-II.

Traditional Chinese herbal medicines are widely accepted as an option for the treatment of colorectal cancers. Danshen (Salviae miltiorrhizae Radix) is widely prescribed in traditional Chinese medicine for cardiovascular diseases. Tanshinone IIA (Tan-IIA) is extracted from Danshen. Our previous studies have shown that Tan-IIA induces apoptosis in Colo205 human colon cancer cells in vitro and in vivo. In the present study, we investigated the efficacy of Tan-IIA and 5-fluorouracil (5-FU) in a Colo205 cell xenograft model. For in vivo studies, SCID mice were engrafted with Colo205 cells and from day 10 onwards were randomly divided into 3 groups and treated with 5-FU plus Tan-IIA, 5-FU plus corn oil, and the vehicle alone. At the end of a 4-week dosing schedule, the SCID mice were sacrificed and xenograft tumors were dissected for protein western blot analysis. Our results showed that the Colo205 xenograft model co-treated with Tan-IIA plus 5-FU caused a reduction in the xenograft tumor volumes and decreased P-glycoprotein (P-gp) and microtubule-associated protein light chain 3 (LC3)-II expression compared to 5-FU alone. Based on these observations, it may be possible to develop Tan-IIA plus 5-FU as therapeutic agents for human colon cancer.

The treatment of glioblastoma multiforme through activation of microglia and TRAIL induced by rAAV2-mediated IL-12 in a syngeneic rat model.

BACKGROUND: Microglial cells are the predominant immune cells in malignant brain tumors, but tumors may release some factors to reduce their defensive functions. Restoration of the anti-cancer function of microglia has been proposed as a treatment modality for glioblastoma. We examined the effect of intra-cranially administered recombinant adeno-associated virus encoding interleukin-12 (rAAV2/IL12) on transfection efficiency, local immune activity and survival in a rat model of glioblastoma multiforme. METHODS: F344 rats were injected with rAAV2/IL12 and implanted with syngeneic RG2 cells (glioblastoma cell line). Intracerebral interleukin-12 and interferon-γ concentrations were determined by ELISA. Activation of microglia was determined by expressions of ED1 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) which were evaluated by Western blotting and immunohistochemistry. The proliferation of cancer cells was evaluated with Ki67 immunohistochemistry and apoptosis of cancer cells with TUNEL. RESULTS: The brains treated with rAAV2/IL-12 maintained high expression of interleukin-12 and interferon-γ for at least two months. In syngeneic tumor model, brains treated with rAAV2/IL12 exhibited more infiltration of activated microglia cells as examined by ED1 and TRAIL stains in the tumor. In addition, the volume of tumor was markedly smaller in AAV2/IL12-treated group and the survival time was significantly longer in this group too. CONCLUSION: The intra-cerebrally administered rAAV2/IL-12 efficiently induces long lasting expression of IL-12, the greater infiltration of activated microglia cells in the tumor associated improved immune reactions, resulting in the inhibited growth of implanted glioblastoma and the increased survival time of these rats.

Tanshinone IIA inhibits BT-20 human breast cancer cell proliferation through increasing caspase 12, GADD153 and phospho-p38 protein expression.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Tanshinone IIA (Tan-IIA) is one of the pure compounds from Salviae miltiorrhizae radix (Danshen). Tan-IIA can inhibit human breast cancer cells but the molecular mechanisms are not well understood. Our previous study showed that Tan-IIA can inhibit hep-J5 human hepatocellular carcinoma cells through the endoplasmic reticulum (ER) stress-induced apoptotic pathway. In the present study, we evaluated the effects of Tan-IIA on BT-20 human breast cancer cells and assessed the involvement of the ER-stress-apoptotic pathway. The cytotoxicity of Tan-IIA in BT-20 cells was measured by the MTT assay. The cell cycles were analyzed by flow cytometry. The expression of ER stress-related proteins in BT-20 cells treated with Tan-IIA were evaluated by western blotting and immunocytochemical staining. These results showed that Tan-IIA can inhibit BT-20 cells and increase the sub-G1 phase in a time- and dose-dependent manner. Tan-IIA could increase the protein expression of caspase 12, GADD153, caspase 3, phospho-JNK, phospho-p38 and Bax, but decreased Bcl-xl and phospho-ERK expression in BT-20 cells. These findings indicate that Tan-IIA possesses therapeutic potential for human breast cancer BT-20 cells; one of the molecular mechanisms may be through inducing ER stress and the MAPK pathway to induce apoptosis and inhibit proliferation.

Tanshinone IIA inhibits human breast cancer MDA-MB-231 cells by decreasing LC3-II, Erb-B2 and NF-κBp65.

The ability of tanshinone IIA (Tan-IIA) to inhibit the proliferation of human breast cancer cell lines in vitro and in vivo is well documented. However, the molecular mechanisms have not been fully elucidated. In the present study, MDA-MB-231 cells were treated with different concentrations of Tan-IIA for 48 h, followed by protein extraction for western blotting. For an in vivo study, MDA-MB-231 cells were implanted directly into female SCID mice which were divided randomly into three groups to be treated with vehicle, Tan-IIA (20 mg/kg) and Tan-IIA (60 mg/kg) every other day orally, with treatment starting 4 weeks after inoculation with the MDA-MB-231 cells. The results showed that Tan-IIA inhibited the proliferation of MDA-MB-231 cells and decreased the protein expression of LC3-II and Erb-B2 in vitro. Treatment with Tan-IIA (20 or 60 mg/kg) for 90 days resulted in a reduction in tumor size and weight compared to the control group. The protein expression of NF-κBp65 was reduced, while caspase-3 was up-regulated compared to the control group. These findings indicate that Tan-IIA inhibits tumor growth in a MDA-MB-231 xenograft animal model. One of the molecular mechanisms may be through a decrease in NF-κBp65 and an increase in caspase-3 expression.

Tanshinone IIA inhibits human hepatocellular carcinoma J5 cell growth by increasing Bax and caspase 3 and decreasing CD31 expression in vivo.

Tanshinone IIA (Tan-IIA) decreases the viability of human hepatocellular carcinoma (HCC) cells through the induction of apoptosis in vitro. However, there are no reports that Tan-IIA is capable of inhibiting J5 HCC cell growth in vivo. In this study, J5 cells were implanted directly into nude SCID mice which were divided randomly into four groups to be treated with vehicle, Tan-IIA (30 mg/-kg of body weight, Q.week days 3 and 5), 5-FU (30 mg/-kg of body weight, Q.week day 1) or Tan-IIA (30 mg/-kg of body weight, Q.week days 3 and 5) plus 5-FU (30 mg/-kg of body weight, Q.week day 1). Each agent was injected intraperitoneally, with treatment starting 4 weeks after inoculation with J5 cells. Treatment with Tan-IIA 30 mg/-kg or with 30 mg/-kg of 5­FU resulted in a reduction in tumor size and weight compared with the control group. The protein expression of Bax and caspase-3 in the J5 xenograft tumors treated with Tan-IIA 30 mg/-kg or with 30 mg/kg of 5-FU was upregulated, whereas that of CD31 was downregulated compared with the control group. These findings indicate that Tan-IIA may inhibit tumor growth in a J5 xenograft animal model by increasing Bax and caspase 3 and decreasing CD31 expression in vivo.