Andrographolide Exhibits Anticancer Activity against Breast Cancer Cells (MCF-7 and MDA-MB-231 Cells) through Suppressing Cell Proliferation and Inducing Cell Apoptosis via Inactivation of ER-α Receptor and PI3K/AKT/mTOR Signaling

Breast cancer is the most common cancer among women worldwide. Chemotherapy followed by endocrine therapy is the standard treatment strategy after surgery or radiotherapy. However, breast cancer is highly resistant to the treatments leading to the recurrence of breast cancer. As a result, the development of alternative medicines derived from natural plants with fewer side effects is being emphasized. Andrographolide isolated from Andrographis paniculata is one of the potential substances with anti-cancer properties in a variety of cell types, including breast cancer cells. This study aims to investigate the anti-cancer effects of andrographolide in breast cancer cells by evaluating cell viability and apoptosis as well as its underlying mechanisms through estrogen receptor (ER)-dependent and PI3K/AKT/mTOR signaling pathways. Cell viability, cell apoptosis, mRNA or miRNA, and protein expression were examined by MTT assay, Annexin V-FITC, qRT-PCR, and Western blot analysis, respectively. MCF-7 and MDA-MB-231 cell viability was reduced in a concentration- and time-dependent manner after andrographolide treatment. Moreover, andrographolide induced cell apoptosis in both MCF-7 and MDA-MB-231 cells by inhibiting Bcl-2 and enhancing Bax expression at both mRNA and protein levels. In MCF-7 cells, the ER-positive breast cancer, andrographolide showed an inhibitory effect on cell proliferation through downregulation of ERα, PI3K, and mTOR expression levels. Andrographolide also inhibited MDA-MB-231 breast cancer cell proliferation via induction of cell apoptosis. However, the inhibition of MCF-7 and MDA-MB-231 cell proliferation of andrographolide treatment did not disrupt miR-21. Our findings showed that andrographolide possesses an anti-estrogenic effect by suppressing cell proliferation in MCF-7 cells. The effects were comparable to those of the anticancer drug fulvestrant in MCF-7 cells. This study provides new insights into the anti-cancer effect of andrographolide on breast cancer and suggests andrographolide as a potential alternative from the natural plant for treating breast cancer types that are resistant to tamoxifen and fulvestrant.     

Hypericin-induced photocytotoxicity is connected with G2/M arrest in HT-29 and S-phase arrest in U937 cells

Susceptibility of the HT-29 human colon adenocarcinoma cell line and human myeloid leukemia cell line U937 to hypericin-mediated photocytotoxicity was investigated and compared in this study. Cellular parameters as viability, cell number, metabolic activity and total protein amount were monitored in screening experiments with subsequent cell-cycle analysis and apoptosis detection to determine the cellular response of the different tumor types to various concentrations of photoactivated hypericin. The results show concentration dependence of the photosensitizer's cytotoxicity on the studied cell lines, with higher sensitivity of U937 cells. Whereas the two extreme hypericin concentrations (1 x 10(-9) M and 1 x 10(-6) M) resulted in similar changes in all tested cellular parameters on the two studied cell lines, 1 x 10(-8) M and 1 x 10(-7) M hypericin treatment resulted in different responses of the cell lines in all monitored parameters except for viability. Although leukemic cells proved sensitive to both 1 x 10(-8) M and 1 x 10(-7) M hypericin, significant changes on HT-29 cells were detected only after the 1 x 10(-7) M hypericin concentration. Cell-cycle arrest was related to simultaneously occurring apoptosis in colon cancer. Remarkable is the difference in cell-cycle profile where G2/M arrest in colon cancer cells versus accumulation of leukemic cells in the S phase appears. This suggests that hypericin treatment affecting the cell-cycle machinery of different cancer cells is not universal in effect.     

Anti-Cancer Effects of α-Cubebenoate Derived from Schisandra chinensis in CT26 Colon Cancer Cells

α-Cubebenoate derived from Schisandra chinensis has been reported to possess anti-allergic, anti-obesity, and anti-inflammatory effects and to exhibit anti-septic activity, but its anti-cancer effects have not been investigated. To examine the anti-cancer activity of α-cubebenoate, we investigated its effects on the proliferation, apoptosis, and metastasis of CT26 cells. The viabilities of CT26 cells (a murine colorectal carcinoma cell line) and HCT116 cells (a human colon cancer cell line) were remarkably and dose-dependently diminished by α-cubebenoate, whereas the viability of CCD-18Co cells (a normal human fibroblast cell line) were unaffected. Furthermore, α-cubebenoate treatment increased the number of apoptotic CT26 cells as compared with Vehicle-treated cells and increased Bax, Bcl-2, Cas-3, and Cleaved Cas-3 protein levels by activating the MAP kinase signaling pathway. α-Cubebenoate also suppressed CT26 migration by regulating the PI3K/AKT signaling pathway. Furthermore, similar reductions were observed in the expression levels of some migration-related proteins including VEGFA, MMP2, and MMP9. Furthermore, reduced VEGFA expression was found to be accompanied by the phosphorylations of FAK and MLC in the downstream signaling pathway of adhesion protein. The results of the present study provide novel evidence that α-cubebenoate can stimulate apoptosis and inhibit metastasis by regulating the MAPK, PI3K/AKT, and FAK/MLC signaling pathways.     

Inhibition of Interleukin-6-Induced Matrix Metalloproteinase-2 Expression and Invasive Ability of Lemon Peel Polyphenol Extract in Human Primary Colon Cancer Cells

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.     

Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells

This study aims to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin (Cur) on leukemic stem cell and leukemic cell lines, including KG-1a and KG-1 (FLT3⁺ LSCs), EoL-1 (FLT3⁺ LCs), and U937 (FLT3⁻ LCs). The cytotoxicity of co-treatments of doxorubicin (Dox) or idarubicin (Ida) at concentrations of the IC₁₀–IC₈₀ values and each concentration of Cur at the IC₂₀, IC₃₀, IC₄₀, and IC₅₀ values (conditions 1, 2, 3, and 4) was determined by MTT assays. Dox–Cur increased cytotoxicity in leukemic cells. Dox–Cur co-treatment showed additive and synergistic effects in several conditions. The effect of this co-treatment on FLT3 expression in KG-1a, KG-1, and EoL-1 cells was examined by Western blotting. Dox–Cur decreased FLT3 protein levels and total cell numbers in all the cell lines in a dose-dependent manner. In summary, this study exhibits a novel report of Dox–Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.     

Free radical scavenging and radioprotective effects of carnosine and anserine

Two histidine-containing natural dipeptides, carnosine and anserine (β-alanyl-1-methyl-l-histidine), have been examined for their antioxidant and radioprotective abilities. Pulse radiolysis studies indicated the antioxidative properties of carnosine and anserine aqueous solutions at different pH. The rate constants for the reaction OH radical with carnosine at neutral pH were determined to be 5.3×10⁹ M⁻¹ s⁻¹ at 300 nm, and 4.1×10⁹ M⁻¹ s⁻¹ at 400 nm, respectively. Carnosine and anserine also protected plasmid pUC18 DNA from X-ray radiation-induced strand breaks as evidenced from the studies by agarose gel electrophoresis. Carnosine showed higher protective efficiency under the experimental conditions. Our data demonstrated that carnosine and anserine may play an important role in the maintenance of the antioxidant system.     

An Approach to Minimize Tumour Proliferation by Reducing the Formation of Components for Cell Membrane

Isoprenoids are natural compounds essential for a great number of cellular functions. One of them is farnesol (FOH), which can reduce cell proliferation, but its low solubility in aqueous solvents limits its possible clinical use as a pharmacological tool. One alternative is the use of cyclodextrins (CDs) which house hydrophobic molecules forming inclusion complexes. To assess FOH potential application in anticancer treatments, Sulfobutylated β-cyclodextrin Sodium Salt (SBE-β-CD) was selected, due to it has high solubility, approbation by the FDA, and numerous studies that ensure its safety to be administered parenterally or orally without nephrotoxicity associated. The therapeutic action of farnesol and complex were studied in different carcinoma cells, compared with a normal cell line. Farnesol showed selectivity, affecting the viability of colon and liver cancer cells more than in breast cancer cells and fibroblasts. All cells suffered apoptosis after being treated with 150 μM of free FOH, but the complex reduced their cell viability between 50 and 75%. Similar results were obtained for both types of isomers, and the addition of phosphatidylcholine reverses this effect. Finally, cell cycle analysis corroborates the action of FOH as inducer of a G0/G1 phase; when the cells were treated using the complex form, this viability was reduced, reaching 50% in the case of colon and liver, 60% in fibroblasts, and only 75% in breast cancer.     

An Insight into the Anti-Angiogenic and Anti-Metastatic Effects of Oridonin: Current Knowledge and Future Potential

Cancer is one of the leading causes of death worldwide, with a mortality rate of more than 9 million deaths reported in 2018. Conventional anti-cancer therapy can greatly improve survival however treatment resistance is still a major problem especially in metastatic disease. Targeted anti-cancer therapy is increasingly used with conventional therapy to improve patients’ outcomes in advanced and metastatic tumors. However, due to the complexity of cancer biology and metastasis, it is urgent to develop new agents and evaluate the anti-cancer efficacy of available treatments. Many phytochemicals from medicinal plants have been reported to possess anti-cancer properties. One such compound is known as oridonin, a bioactive component of Rabdosia rubescens. Several studies have demonstrated that oridonin inhibits angiogenesis in various types of cancer, including breast, pancreatic, lung, colon and skin cancer. Oridonin’s anti-cancer effects are mediated through the modulation of several signaling pathways which include upregulation of oncogenes and pro-angiogenic growth factors. Furthermore, oridonin also inhibits cell migration, invasion and metastasis via suppressing epithelial-to-mesenchymal transition and blocking downstream signaling targets in the cancer metastasis process. This review summarizes the recent applications of oridonin as an anti-angiogenic and anti-metastatic drug both in vitro and in vivo, and its potential mechanisms of action.     

Anticancer Effects and Molecular Action of 7-α-Hydroxyfrullanolide in G2/M-Phase Arrest and Apoptosis in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression. TNBC cells respond poorly to targeted chemotherapies currently in use and the mortality rate of TNBC remains high. Therefore, it is necessary to identify new chemotherapeutic agents for TNBC. In this study, the anti-cancer effects of 7-α-hydroxyfrullanolide (7HF), derived from Grangea maderaspatana, on MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells were assessed using MTT assay. The mode of action of 7HF in TNBC cells treated with 6, 12 and 24 µM of 7HF was determined by flow cytometry and propidium iodide (PI) staining for cell cycle analysis and annexin V/fluorescein isothiocyanate + PI staining for detecting apoptosis. The molecular mechanism of action of 7HF in TNBC cells was investigated by evaluating protein expression using proteomic techniques and western blotting. Subsequently, 7HF exhibited the strongest anti-TNBC activity toward MDA-MB-468 cells and a concomitantly weak toxicity toward normal breast cells. The molecular mechanism of action of low-dose 7HF in TNBC cells primarily involved G2/M-phase arrest through upregulation of the expression of Bub3, cyclin B1, phosphorylated Cdk1 (Tyr 15) and p53-independent p21. Contrastingly, the upregulation of PP2A-A subunit expression may have modulated the suppression of various cell survival proteins such as p-Akt (Ser 473), FoxO3a and β-catenin. The concurrent apoptotic effect of 7HF on the treated cells was mediated via both intrinsic and extrinsic modes through the upregulation of Bax and active cleaved caspase-7–9 expression and downregulation of Bcl-2 and full-length caspase-7–9 expression. Notably, the proteomic approach revealed the upregulation of the expression of pivotal protein clusters associated with G1/S-phase arrest, G2/M-phase transition and apoptosis. Thus, 7HF exhibits promising anti-TNBC activity and at a low dose, it modulates signal transduction associated with G2/M-phase arrest and apoptosis.     

Entelon® (Vitis vinifera Seed Extract) Prevents Cancer Metastasis via the Downregulation of Interleukin-1 Alpha in Triple-Negative Breast Cancer Cells

Interleukin-1 (IL1) is a proinflammatory cytokine and promotes cancer cell proliferation and invasiveness in a diversity of cancers, such as breast and colon cancer. Here, we focused on the pharmacological effect of Entelon^® (ETL) on the tumorigenesis of triple-negative breast cancer (TNBC) cells by IL1-alpha (IL1A). IL1A enhanced the cell growth and invasiveness of TNBC cells. We observed that abnormal IL1A induction is related with the poor prognosis of TNBC patients. IL1A also increased a variety of chemokines such as CCL2 and IL8. Interestingly, IL1A expression was reduced by the ETL treatment. Here, we found that ETL significantly decreased the MEK/ERK signaling pathway in TNBC cells. IL1A expression was reduced by UO126. Lastly, we studied the effect of ETL on the metastatic potential of TNBC cells. Our results showed that ETL significantly reduced the lung metastasis of TNBC cells. Our results showed that IL1A expression was regulated by the MEK/ERK- and PI3K/AKT-dependent pathway. Taken together, ETL inhibited the MEK/ERK and PI3K/AKT signaling pathway and suppressing the lung metastasis of TNBC cells through downregulation of IL1A. Therefore, we propose the possibility of ETL as an effective adjuvant for treating TNBC.     

AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2^-/- cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin-mediated cancer control.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.     

Ethiopian Medicinal Plants Traditionally Used for the Treatment of Cancer; Part 3: Selective Cytotoxic Activity of 22 Plants against Human Cancer Cell Lines

Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC₅₀ values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC₅₀ values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.     

Component Analysis and Anti-Colorectal Cancer Mechanism via AKT/mTOR Signalling Pathway of Sanghuangporus vaninii Extracts

Sanghuangporus vaninii (Ljub.) L.W. Zhou & Y.C. Dai (SV) is a major cultivar of Sanghuang, which is well known as an excellent anti-tumour drug and reaches the mainstream market in China. Water, 60% ethanol and 95% ethanol were used to extract the drug, and three kinds of polar extracts were obtained separately. Compared with water extracts and 95% ethanol extracts, the 60% ethanol extract had the highest flavonoid content, and its polysaccharide content was greater than that in the 95% ethanol extract and lower than that in the water extract. Its essential components were phenolics whose majority were phenolic acids, flavonoids and phenylpropanoids. This extract has better inhibition effects on the proliferation of SW480 human colon cancer cells, inducing cell apoptosis and blocking G2/M period cells. It can significantly inhibit gene expression and reduce the activation of the AKT/mTOR signalling pathway. The anti-cancer activity of the 60% ethanol extract is satisfactory and may be a result of the combined effects of polysaccharides and flavonoids. The data suggest that the 60% ethanol extract can be used as an adjuvant for chemotherapy and as a potential anti-cancer agent with broad development prospects.     

Palmatine,a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica,Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER⁺/HER2⁻ breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC₅₀ values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.     

Carnosine to Combat Novel Coronavirus (nCoV): Molecular Docking and Modeling to Cocrystallized Host Angiotensin-Converting Enzyme 2 (ACE2) and Viral Spike Protein

Aims: Angiotensin-converting enzyme 2 (ACE2) plays an important role in the entry of coronaviruses into host cells. The current paper described how carnosine, a naturally occurring supplement, can be an effective drug candidate for coronavirus disease (COVID-19) on the basis of molecular docking and modeling to host ACE2 cocrystallized with nCoV spike protein. Methods: First, the starting point was ACE2 inhibitors and their structure–activity relationship (SAR). Next, chemical similarity (or diversity) and PubMed searches made it possible to repurpose and assess approved or experimental drugs for COVID-19. Parallel, at all stages, the authors performed bioactivity scoring to assess potential repurposed inhibitors at ACE2. Finally, investigators performed molecular docking and modeling of the identified drug candidate to host ACE2 with nCoV spike protein. Results: Carnosine emerged as the best-known drug candidate to match ACE2 inhibitor structure. Preliminary docking was more optimal to ACE2 than the known typical angiotensin-converting enzyme 1 (ACE1) inhibitor (enalapril) and quite comparable to known or presumed ACE2 inhibitors. Viral spike protein elements binding to ACE2 were retained in the best carnosine pose in SwissDock at 1.75 Angstroms. Out of the three main areas of attachment expected to the protein–protein structure, carnosine bound with higher affinity to two compared to the known ACE2 active site. LibDock score was 92.40 for site 3, 90.88 for site 1, and inside the active site 85.49. Conclusion: Carnosine has promising inhibitory interactions with host ACE2 and nCoV spike protein and hence could offer a potential mitigating effect against the current COVID-19 pandemic.     

Triptolide-induced suppression of phospholipase D expression inhibits proliferation of MDA-MB-231 breast cancer cells

In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-µM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFκB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFκB activation and then decreases cell proliferation.