Comprehensive fluorogenic derivatization–liquid chromatography/tandem mass spectrometry proteomic analysis of colorectal cancer cell to identify biomarker candidate

Existing colorectal cancer biomarkers are insufficient for providing a quick and accurate diagnosis, which is critical for a good prognosis. More appropriate biomarkers are thus needed. To identify new colorectal cancer biomarker candidates, we conducted a comprehensive differential proteomic analysis of six cancer cell lines and a normal cell line, utilizing a fluorogenic derivatization–liquid chromatography–tandem mass spectrometry (FD‐LC‐MS/MS) approach. Two sets of intracellular biomarker candidates were identified: one for colorectal cancer, and the other for metastatic colorectal cancer. Our results suggest that cooperative expression of FABP5 and cyclophilin A might be linked to Her2 signaling. Upregulation of LDHB and downregulation of GAPDH suggest the existence of a specific nonglycolytic energy production pathway in metastatic colorectal cancer cells. Downregulation of 14‐3‐3ζ/δ, cystatin‐B, Ran and thioredoxin could be a result of their secretion, which then stimulates metastasis via activity in the sera and ascitic fluids. We propose a possible flow scheme to describe the dynamics of protein expression in colorectal cancer cells leading to tumor progression and metastasis via cell proliferation, angiogenesis, disorganization of actin filaments and epithelial–mesenchymal transition. Our results suggest that colorectal tumor progression may be regulated by signaling mediated by Her2, hypoxia, and TGFβ. Copyright © 2012 John Wiley & Sons, Ltd.     

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.     

Analysis of polyamines as carbamoyl derivatives in urine and serum by liquid chromatography-tandem mass spectrometry

A quantitative analysis of polyamines in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The polyamines were carbamylated with isobutyl chloroformate, extracted with diethyl ether under pH 9.0, and analyzed by LC-MS/MS with single reaction monitoring mode. The limit of quantification was 1 ng/mL based on a signal-to-noise ratio>3, and the correlation coefficient (r2) for the calibration curves was >0.99 for both urine and serum samples. The present method was applied to urine and serum samples from 30 breast cancer patients and 30 normal female controls. There was no significant difference in the urinary polyamine levels between breast cancer patients and controls. However, 1,3-diaminopropane, putrescine, spermine and N-acetylspermidine levels in serum increased in breast cancer patients. These four serum polyamines may be a good index to study both production and metabolism of polyamines, and a useful tool in assessment of the polyamine status of breast cancer patients.     

5‐Methoxytryptophan‐dependent inhibition of oral squamous cell carcinoma metastasis

The metastatic status of oral cancer is highly associated with the overall survival rate of patients. Previous studies have revealed that the endogenous tryptophan metabolite 5‐methoxytryptophan (5‐MTP) can downregulate cyclooxygenase‐2 expression; suppress tumor proliferation, migration, and invasion; and reduce the tumor size. To improve the understanding of the molecular mechanisms involved in the regulation of 5‐MTP in the tumorigenesis of oral cancer, we conducted a comparative wound healing and transwell invasion assays. Our results revealed that 5‐MTP reduce oral cancer cell migration and invasion ability. In addition, the results of an in vivo assay demonstrated that the growth of primary tumors was significantly inhibited by 5‐MTP in OC3 oral cancer cells and in invasive OC3‐I5 oral cancer cells. Moreover, enlarged spleens were observed in OC3‐I5‐implanted severe combined immunodeficiency mice although 5‐MTP can inhibit spleen enlargement. Through comparative proteomics, we identified 32 differentially regulated protein spots by using 2D‐DIGE/MALDI‐TOF MS analyses. Some of the differentially regulated proteins such as amadillo‐repeat‐containing X‐linked protein 1, phosphoglycerate kinase 1, tropomyosin alpha‐1, and tropomyosin alpha‐4 may be associated with the 5‐MTP‐dependent inhibition of oral cancer growth and metastasis. We conclude that 5‐MTP plays a crucial role in inhibiting in vitro and in vivo cancer invasion and metastasis.     

Conjugation of Palbociclib with MHI-148 Has an Increased Cytotoxic Effect for Breast Cancer Cells and an Altered Mechanism of Action

The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor-positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive, triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative, as well as estrogen receptor-positive, breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells, suggesting its mechanism of action differs from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds, as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.     

Serological proteome analysis of dogs with breast cancer unveils common serum biomarkers with human counterparts

Canine mammary tumor is being touted as a model for investigating the human breast cancer. Breast cancer of the both species has similar biological behavior, histopathologic characteristics, and metastatic pattern. In this study, we used the serological proteome analysis to detect autoantigens that elicit a humoral response in dogs with mammary tumor in order to identify serum biomarkers with potential usefulness as diagnostic markers and to better understand molecular mechanisms underlying canine breast cancer development. Protein extract from a cell line was subject to 2DE followed by Western blotting using sera from 15 dogs with mammary tumor and sera from 15 healthy control dogs. Immunoreactive autoantigens were subsequently identified by the MALDI‐TOF MS. Four autoantigens, including manganese‐superoxide dismutase, triose phosphate isomerase, alpha‐enolase, and phosphoglycerate mutase1, with significantly higher immunoreactivity in the tumor samples than in the normal samples were identified as biomarker candidates. Immunohistochemistry and Western blotting revealed higher expression of these biomarkers in the malignant tumors than in the normal or benign tumors. The autoantigens found in this study have been reported to elicit autoantibody response in the human breast cancer, indicating the similarity of breast cancer proteome profile in dogs with that in human beings.     

Determination of single cell surface protein expression using a tagless microfluidic method

We describe a method to detect the expression of a surface protein in single cells without prior labeling or manipulation using a microfluidic device. When the protein is expressed on a cell surface, it undergoes transient bond formation with an immobilized ligand as the cell is pumped through a microfluidic channel, resulting in a specific decrease in the cell's velocity. We were able to detect the expression of interleukin 13 receptor alpha 2 (IL13Rα2) differentially expressed on LM2 cells, a subline of MDA-MB-231 human breast cancer cells with a unique lung metastatic capability. The detection of cells with high expression of the protein was near 100% sensitive and 100% specific. We also provided proof of principle of multiplexing by tracking the same cell over two, differentially-coated patches. The method is non-destructive and cells can be collected for reanalysis. The system can identify positive cells in a cell mixture. This method will have a potential impact in analyzing cancer cells when only a few are available, such as the case with needle aspirates and when labeling and manipulation result in cell loss.     

A functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin- β1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral- MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.     

Inhibition of NF-kB/IL-6/JAK2/STAT3 Pathway and Epithelial-Mesenchymal Transition in Breast Cancer Cells by Azilsartan

Metastatic breast cancer is an incurable form of breast cancer that exhibits high levels of epithelial-mesenchymal transition (EMT) markers. Angiotensin II has been linked to various signaling pathways involved in tumor cell growth and metastasis. The aim of this study is to investigate, for the first time, the anti-proliferative activity of azilsartan, an angiotensin II receptor blocker, against breast cancer cell lines MCF-7 and MDA-MB-231 at the molecular level. Cell viability, cell cycle, apoptosis, colony formation, and cell migration assays were performed. RT-PCR and western blotting analysis were used to explain the molecular mechanism. Azilsartan significantly decreased the cancer cells survival, induced apoptosis and cell cycle arrest, and inhibited colony formation and cell migration abilities. Furthermore, azilsartan reduced the mRNA levels of NF-kB, TWIST, SNAIL, SLUG and bcl2, and increased the mRNA level of bax. Additionally, azilsartan inhibited the expression of IL-6, JAK2, STAT3, MMP9 and bcl2 proteins, and increased the expression of bax, c-PARP and cleaved caspase 3 protein. Interestingly, it reduced the in vivo metastatic capacity of MDA-MBA-231 breast cancer cells. In conclusion, the present study revealed, for the first time, the anti-proliferative, apoptotic, anti-migration and EMT inhibition activities of azilsartan against breast cancer cells through modulating NF-kB/IL-6/JAK2/STAT3/MMP9, TWIST/SNAIL/SLUG and apoptosis signaling pathways.     

乳腺癌代谢物组模式特征发现方法及HPLC/M S/M S分析

提出一种基于单独最优特征组合和BP神经网络的代谢物组模式特征发现方法,并用其寻找到尿样中与乳腺癌最为相关的4种核苷,组成一组特异性检测参数.经HPLC/MS/MS联用法鉴定,它们是乳清酸核苷、1-甲酰化腺苷、S-腺苷-L-蛋氨酸及N²-甲酰化鸟苷.将这4种核苷作为输入变量,用BP神经分类网络建立乳腺癌诊断模型.留一法交叉验证和独立验证结果表明,该模型预测准确率达到90%以上.     

Gypensapogenin H from hydrolyzate of total Gynostemma pentaphyllum saponins induces apoptosis in human breast carcinoma cells

Abstract

Gypensapogenin H (Gyp H) is a novel dammarane-type triterpene, isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum. Our previous work demonstrated that Gyp H exhibited potent growth inhibitory effects on tumor cells. It significantly inhibited the growth of human breast cancer cells (MDA-MB-231), while having low toxicity to normal human breast epithelial cells, MCF-10a. Further mechanistic study demonstrated that Gyp H decreased survival, inhibited proliferation, migration, induced apoptosis and led to cell cycle arrest. For the MDA-MB-231 cell lines, Gyp H increased expression of P21, Bax and cytochrome c, induced PARP cleavage and activated caspases. Gyp H also reduced expression of CDK2/4, CyclinD1, E2F1 and Bcl2, which associated with the cell cycle arrest. Thus, our finding may be useful for understanding the mechanism of action of Gyp H on breast cancer cells and suggest that Gyp H would be a leading agent for the treatment of breast cancer.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.     

Application of organometallic chemistry in the formulation of a modern therapeutic drug by Ag nanoparticles green-mediated by Allium to treat the breast cancer

In the recent study, we decided to survey the capacities of metallic nanoparticles formulated by Allium monanthum (AgNPs) as a novel chemotherapeutic drug in the treatment of several types of breast cancers. Characterization of AgNPs was done by UV–Visible Spectroscopy (UV–Vis), Fourier Transformed Infrared Spectroscopy (FT‐IR), Transmission Electron Microscopy (TEM), and Field Emission Scanning Electron Microscopy (FE‐SEM). For investigating the antioxidant properties of AgNO₃, Allium monanthum, and AgNPs, the DPPH test was used in the presence of butylated hydroxytoluene as the positive control. To survey the cytotoxicity and anti-breast cancer effects of AgNO₃, Allium monanthum, and AgNPs, MTT assay was used on the breast adenocarcinoma (MCF7), breast carcinoma (Hs 578Bst), infiltrating ductal cell carcinoma (Hs 319.T), infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines. DPPH test revealed similar antioxidant potentials for Allium monanthum, AgNPs, and butylated hydroxytoluene. Silver nanoparticles had very low cell viability and anti-breast cancer properties dose-dependently against MCF7, Hs 578Bst, Hs 319.T, UACC-3133, UACC-732, and MDA-MB-453 cell lines without any cytotoxicity on the normal cell line. The best result of anti-breast cancer properties of AgNPs against the above cell lines was seen in the case of the UACC-3133 cell line. According to the above findings, the silver nanoparticles containing Allium monanthum aqueous extract can be administrated in humans for the treatment of several types of breast cancer especially breast adenocarcinoma, breast carcinoma, infiltrating ductal cell carcinoma, infiltrating lobular carcinoma of breast, inflammatory carcinoma of the breast, and metastatic carcinoma.     

基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用

磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti⁴⁺)螯合IMAC材料(Ti⁴⁺-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti⁴⁺-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti⁴⁺-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti⁴⁺-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

Exploration of Potential Tumor Markers for Lung Adenocarcinomas by Two‐Dimensional Gel Electrophoresis Coupled with Nano‐LC/MS/MS

To explore the potential tumor markers for lung adenocarcinoma, two‐dimensional gel electrophoresis (2DE) coupled with nano‐LC/MS/MS was used to analyze the differentially expressed proteins in 10 surgical resected lung adenocarcinoma tissues. 16 proteins were significantly different between the cancer tissue and adjacent normal tissue. Galectin‐1, peroxiredoxin II (Prx II), proapolipoprotein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), aldolase A, enolase 1, neuropolypeptide h3, Prx V, cyclophilin A, vimentin, protein disulfide isomerase (PDI), tropomyosin 3 (TPM 3), glutathione S‐transferase Pi (GST‐Pi), manganese superoxide dismutase (MnSOD), and cofilin 1 were up‐regulated in the cancer tissue. On the other hand, profilin was down‐regulated in the cancer tissue. Among these proteins, six proteins were validated by Western blot analysis. The identified proteins contributing to the spectrum of cancer progression may be used as potential diagnostic biomarkers for lung adenocarcinomas.     

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.     

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.