Expression and Characterization of a Novel Recombinant Cytotoxin II from Naja naja oxiana Venom: A Potential Treatment for Breast Cancer

Breast cancer (BC) is among the leading causes of mortality from cancer in women. Many of the available anticancer drugs have various side effects. Therefore, researchers are seeking novel anticancer agents particularly from natural compounds and in this regard, snake venom is still one of the main sources of drug discovery. Previous studies showed potential anticancer effects of Cytotoxin II (CTII) from Naja naja oxiana against the different types of cancers. In this study, a pET-SUMO-CTII vector was transformed into SHuffle® T7 Express, an Escherichia coli strain, for recombinant protein expression (rCTII) and the cytotoxic effects of this protein was assessed in MCF-7 cells. The flow cytometry assay was applied to measure the apoptosis and cell cycle. Also, mRNA levels of the Bax, Bcl2, P53, caspase-3, caspase-8, caspase-9, caspase-10, matrix metalloproteinases (MMP)-3, and MMP-9 were analyzed by quantitative real-time PCR to determine the underlying cellular pathways affected by rCTII. The results of this study showed that treatment with 4 µg mL-1 of rCTII enhanced apoptosis through the intrinsic and extrinsic pathways. Also, the increase of the cells' proportion in the sub-G1 phase as well as a reduction in S phase was observed. In addition, the expression of MMP-3 and MMP-9 was decreased in the treated group in comparison to the control group that may contribute to the reduced migratory ability of tumor cells. These experimental results indicate that rCTII has anti-proliferative potential, and so this protein could be a potential drug for BC therapy in combination with other drugs.

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Mummy Induces Apoptosis Through Inhibiting of Epithelial-Mesenchymal Transition (EMT) in Human Breast Cancer Cells

Galen Medical Journal ◽

10.31661/gmj.v9i0.1812 ◽

2020 ◽

Vol 9 ◽

pp. 1812

Author(s):

Solmaz Rahmani Barouji ◽

Arman Shahabi ◽

Mohammadali Torbati ◽

Seyyed Mohammad Bagher Fazljou ◽

Ahmad Yari Khosroushahi

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Anticancer Activity ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Epithelial Mesenchymal Transition ◽

Control Group ◽

Mrna Levels ◽

Mesenchymal Transition ◽

Mcf 7

Background: Mummy (Iranian pure shilajit) is a remedy with possessing anti-inflammatory, antioxidant and anticancer activities. This study aimed to examine mummy effects on epithelial-mesenchymal transition (EMT) and invasiveness of MCF-7 and MDA-MB-231 breast cancer (BC) cell lines with underlying its mechanism. Materials and Methods: The dose-dependent inhibitory effect of the mummy on cell proliferation in vitro was determined using the MTT assay. Flow cytometry and 4’,6-diamidino-2-phenylindole dihydrochloride staining were respectively used for quantitative and qualitative analysis of cellular apoptosis, and gene expression analysis was conducted using real-time PCR. Results: MDA-MB-231 showed more sensitivity than the MCF-7 cell line to the anticancer activity of mummy, while mummy did not exhibit significant cell cytotoxicity against human normal cells (MCF-10A). The gene expression profile demonstrated a significant decrease in TGF-β1, TGF-βR1, TWIST1, NOTCH1, CTNNB1, SRC along with an increase in E-cadherin mRNA levels in mummy treated cells compared to the untreated control group (P≤0.05). Conclusion: Mummy triggers inhibition of EMT and metastasis in breast cancer cells mainly through the downregulation of TGFβ1 activity, and more studies required to find its specific anticancer activity with details. [GMJ.2020;9:e1812]

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Ductal tree ablation by local delivery of ethanol prevents tumor formation in an aggressive mouse model of breast cancer

Breast Cancer Research ◽

10.1186/s13058-019-1217-x ◽

2019 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Elizabeth Kenyon ◽

Jennifer J. Westerhuis ◽

Maximilian Volk ◽

Jeremy Hix ◽

Shatadru Chakravarty ◽

...

Keyword(s):

Breast Cancer ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Prophylactic Mastectomy ◽

Treated Group ◽

Tumor Formation ◽

Mammary Epithelial ◽

Tumor Incidence ◽

Control Group ◽

Untreated Control Group

Abstract Background Prophylactic mastectomy is the most effective intervention to prevent breast cancer. However, this major surgery has life-changing consequences at the physical, emotional, psychological, and social levels. Therefore, only high-risk individuals consider this aggressive procedure, which completely removes the mammary epithelial cells from which breast cancer arises along with surrounding tissue. Here, we seek to develop a minimally invasive procedure as an alternative to prophylactic mastectomy by intraductal (ID) delivery of a cell-killing solution that locally ablates the mammary epithelial cells before they become malignant. Methods After ID injection of a 70% ethanol-containing solution in FVB/NJ female animals, ex vivo dual stained whole-mount tissue analysis and in vivo X-ray microcomputed tomography imaging were used to visualize ductal tree filling, and histological and multiplex immunohistochemical assays were used to characterize ablative effects and quantitate the number of intact epithelial cells and stroma. After ID injection of 70% ethanol or other solutions in cancer-prone FVB-Tg-C3(1)-TAg female animals, mammary glands were palpated weekly to establish tumor latency and examined after necropsy to record tumor incidence. Statistical difference in median tumor latency and tumor incidence between experimental groups was analyzed by log-rank test and logistic mixed-effects model, respectively. Results We report that ID injection of 70% ethanol effectively ablates the mammary epithelia with limited collateral damage to surrounding stroma and vasculature in the murine ductal tree. ID injection of 70% ethanol into the mammary glands of the C3(1)-TAg multifocal breast cancer model significantly delayed tumor formation (median latency of 150 days in the untreated control group [n = 25] vs. 217 days in the ethanol-treated group [n = 13], p value < 0.0001) and reduced tumor incidence (34% of glands with tumors [85 of 250] in the untreated control group vs. 7.3% of glands with tumor [7 of 95] in the ethanol-treated group, risk ratio = 4.76 [95% CI 1.89 to 11.97, p value < 0.0001]). Conclusions This preclinical study demonstrates the feasibility of local ductal tree ablation as a novel strategy for primary prevention of breast cancer. Given the existing clinical uses of ethanol, ethanol-based ablation protocols could be readily implemented in first-in-human clinical trials for high-risk individuals.

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Efficacy, toxicity, and applicability of high-dose sequential chemotherapy as adjuvant treatment in operable breast cancer with 10 or more involved axillary nodes: five-year results.

Journal of Clinical Oncology ◽

10.1200/jco.1997.15.6.2312 ◽

1997 ◽

Vol 15 (6) ◽

pp. 2312-2321 ◽

Cited By ~ 130

Author(s):

A M Gianni ◽

S Siena ◽

M Bregni ◽

M Di Nicola ◽

S Orefice ◽

...

Keyword(s):

Breast Cancer ◽

Standard Dose ◽

Treated Group ◽

Multicenter Trial ◽

Control Group ◽

High Dose ◽

Axillary Nodes ◽

Major Toxicity ◽

Nonhematologic Toxicity

PURPOSE To assess the efficacy, toxicity, and applicability of high-dose therapy administered as adjuvant initial treatment to women with breast cancer with extensive nodal involvement. PATIENTS AND METHODS Sixty-seven patients with stage II to III breast cancer involving > or = 10 axillary nodes received a novel high-dose sequential (HDS) regimen, including the high-dose administration of three non-cross-resistant drugs (cyclophosphamide, methotrexate, and melphalan) given within the shortest interval of time as possible with hematologic and nonhematologic toxicity. RESULTS Sixty-three patients completed the program as planned, one patient died of acute toxicity, and three patients were switched to standard-dose adjuvant therapy. After a median follow-up duration of 48.5 months and a lead follow-up of 78 months, actuarial relapse-free survival for all 67 registered patients is 57% and overall survival is 70%, respectively. Comparison with a historical control group of 58 consecutive patients showed a significantly superior rate of freedom from relapse for the HDS-treated group (57% v 41%, respectively), in particular when two subgroups of patients, more homogeneous for their number of involved nodes, were compared (65% v 42%). Overall, treatment was of short duration (median, 70 days), required a median of 32 days of hospital stay, and was associated with only a few severe side effects (the most distressing being oral mucositis after melphalan therapy). CONCLUSION HDS therapy emerges as an effective and applicable regimen, whose major toxicity was occasional. Final assessment of its value in a randomized, multicenter trial is presently underway.

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Regulation of Flowering Time by Improving Leaf Health Markers and Expansion by Salicylic Acid Treatment: A New Approach to Induce Flowering in Malus domestica

Frontiers in Plant Science ◽

10.3389/fpls.2021.655974 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kamran Shah ◽

Na An ◽

Svetlana Kamanova ◽

Lijuan Chen ◽

Peng Jia ◽

...

Keyword(s):

Salicylic Acid ◽

Floral Induction ◽

Treated Group ◽

Flower Induction ◽

Control Group ◽

Mrna Levels ◽

Enzymatic Antioxidants ◽

Stress Markers ◽

Health Related ◽

Induction Stage

In the external coincidence model, internal and external molecular signals, provided by the circadian clock and sunlight, respectively, are required to induce flowering. Salicylic acid (SA) applications during floral induction have multiple effects. In the current study, Malus × domestica plants were exposed to SA during the flower-induction stage to analyze the effect on various health markers and flowering. A total of 56 equal-sized Fuji/M9 trees that were about 7 years old were randomly divided into two groups. The first group (SA-treated) was sprayed with 4 mM SA solution, while the second group was sprayed with distilled water which served as control (CK). The SA applications increased various leaf pigments. Abiotic stress markers were increased in CK during the flower-induction stage. In the SA-treated group, non-enzymatic antioxidants increased, whereas in the control group, enzymatic antioxidants increased during the flower-induction stage. Histo-morphometric properties of leaves were significantly improved in the SA-treated group. The relative expression of the mRNA levels of MdMED80, −81, −3, and −41 were significantly increased in SA-treated leaves, leading to an early and increased flowering phenotype. Thus, SA increased leaf expansion and health-related marker levels, which lead to early induction of flowering in M. domestica. Overall, our work established a role for leaf health assessments in the regulation of flowering in M. domestica.

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Agriophyllum Oligosaccharides Ameliorate Diabetic Insulin Resistance Through INS-R/IRS/Glut4-Mediated Insulin Pathway in db/db Mice and MIN6 Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2021.656220 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shuyin Bao ◽

Xiuzhi Wang ◽

Sung Bo Cho ◽

Yan-Ling Wu ◽

Chengxi Wei ◽

...

Keyword(s):

Insulin Resistance ◽

Insulin Receptor ◽

Treated Group ◽

Control Group ◽

Spectrometric Analysis ◽

Precolumn Derivatization ◽

Mrna Levels ◽

Insulin Pathway ◽

Min6 Cells

We have previously reported that Agriophyllum oligosaccharides (AOS) significantly enhance glycemic control by increasing the activation of insulin receptor (INS-R), insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), peroxisome proliferator-activated receptor (PPAR)-γ, and glucose transporter 4 (Glut4) proteins in hepatic tissues. However, the effect of glucose control by AOS on the regulation of pancreatic tissues in db/db mice and MIN6 cells remains to be determined. An oral dose of AOS (380 or 750 mg/kg) was administered to type-2 diabetic db/db mice for 8 weeks to determine whether AOS regulates glucose by the INS-R/IRS/Glut4-mediated insulin pathway. Meanwhile, the effects of AOS on glucose uptake and its related signaling pathway in MIN6 cells were also investigated. The results showed that the random blood glucose (RBG) level in the AOS-treated group was lower than that in the control group. AOS reduced the levels of glycated hemoglobin (HbA1c) and free fatty acid (FFA) and significantly improved the pathological changes in the pancreatic tissues in db/db mice. Moreover, immunohistochemical analysis revealed that the expression of INS-R, IRS-1, IRS-2, and Glut4 was increased in the AOS-treated group than in the model group. Further, in vitro experiments using MIN6 cells showed that AOS regulated INS-R, IRS-1, IRS-2, and Glut4 protein and mRNA levels and attenuated insulin resistance and cell apoptosis. The results of both in vitro and in vivo experiments were comparable. Ultra-performance liquid chromatography coupled with time-of-flight mass spectrometric analysis of AOS with precolumn derivatization with 3-amino-9-ethylcarbazole (AEC) tentatively identified five types of sugars: glucose, lactose, rutinose, glucuronic acid, and maltotriose. Our present study clearly showed that AOS is efficacious in preventing hyperglycemia, possibly by increasing insulin sensitivity and improving IR by regulating the INS-R/IRS/Glut4 insulin signal pathway. Therefore, AOS may be considered as a potential drug for diabetes treatment.

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Cytoplasm lipids can be modulated through hormone-sensitive lipase and are related to mitochondrial function in porcine IVM oocytes

Reproduction Fertility and Development ◽

10.1071/rd19047 ◽

2020 ◽

Vol 32 (7) ◽

pp. 667

Author(s):

Qingrui Zhuan ◽

Haojia Ma ◽

Jing Chen ◽

Yuxi Luo ◽

Yan Luo ◽

...

Keyword(s):

Oocyte Maturation ◽

Mitochondrial Function ◽

Polar Body ◽

Treated Group ◽

Hormone Sensitive Lipase ◽

Control Group ◽

Intracellular Camp ◽

Mrna Levels ◽

Negative Effects ◽

Hormone Sensitive

Intracellular lipids provide energy for oocyte maturation and development. Triglycerides are the main components of cytoplasm lipid droplets, and hydrolysis of triglycerides requires several lipase-mediated steps. The aim of this study was to determine the effects of the β-adrenoceptor agonist isoproterenol (ISO) and the hormone-sensitive lipase (HSL) inhibitor CAY10499 on the IVM of porcine oocytes. ISO (5mg L−1) and CAY10499 (20mg L−1) had positive and negative effects respectively on invitro oocyte maturation and subsequent embryo development. The rates of polar body extrusion, cleavage and blastocyst formation were significantly higher in the ISO-treated group than the control and CAY10499-treated groups. ISO treatment also upregulated intracellular cAMP levels in comparison with the control group, while CAY10499 significantly increased the triglyceride content of matured oocytes when compared with other groups, consistent with the observed decrease in LIPE (HSL) mRNA levels. Furthermore, the inhibitory effects of CAY10499 included decreases in mitochondrial membrane potential and mitochondrial temperature. These results indicate that ISO has a positive effect on the IVM of porcine oocytes, and that intracellular lipid metabolism can be modulated by CAY10499 through inhibition of HSL and is closely related to mitochondrial function.

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Measuring functional HER2-driven signaling status ex vivo to predict response to HER2 therapy: Results from a mouse breast tumor xenograft study.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23203 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23203-e23203

Author(s):

Lance G. Laing ◽

David J Burns ◽

Yao Huang ◽

Ian A. MacNeil ◽

Benjamin E. Rich ◽

...

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Breast Tumor ◽

Treated Group ◽

Tumor Xenograft ◽

Control Group ◽

Her2 Expression ◽

Her2 Receptor ◽

Her2 Signaling ◽

Signaling Activity

e23203 Background: Clinical trials indicate a weak correlation between HER2 expression levels and HER2 targeted therapy benefit. Other biological factors, such as HER2 signaling activity, may be important to measure when identifying patients for treatment with HER2 therapies. To measure the HER2 signaling activity of a patient’s live tumor cells, a new assay using an impedance biosensor, the CELx HER2 Signaling Function (CELx HSF) Test, was developed. In this study, we evaluated whether functional HER2 signaling status was more predictive of response to a dual-HER2 kinase inhibitor than HER2 receptor or gene amplification status, using breast tumor xenograft in vivo models. Methods: Two breast cancer cell lines were studied: HCC1954, a HER2+ cell line with normal HER2 signaling according to the CELx HSF Test, and BT483, a HER2- cell line with abnormally high HER2 signaling according to the CELx HSF Test. Thus, the HER2 receptor status of each cell line was opposite its HER2 signaling status. For each cell line, twenty 4-5 week old female NSG mice were injected with two million cells. Mice were randomly assigned to either a control group that received Captisol or a treatment group that received lapatinib at a dose of 20mg/kg daily for 16 days. Results: There was no significant difference in tumor volume between the control and lapatinib-treated groups in the mice injected with the HCC1954 (HER2+, normal HER2 signaling) cells. Average tumor sizes reached 1028.7 ± 166.9 mm3 for the control group and 893.8 ± 111.5 mm3 for the lapatinib-treated group (P = 0.285) by the end of the study. With the BT483 (HER2-, abnormal HER2 signaling) cells, lapatinib treatment significantly slowed down the increase in tumor size. Average tumor sizes reached 328.3 ± 54.9 mm3 for the control group and 192.4 ± 19.4 mm3 for the lapatinib-treated group (P = 0.0049). Conclusions: The results demonstrate that functional HER2-driven signaling status in live tumor cells is more correlative to response to lapatinib in mouse xenograft tumors than HER2 expression level. These findings provide strong evidence that HER2- breast cancer patients with abnormal HER2 signaling may respond to anti-HER2 therapies.

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Effects of Puerariae Flos and Puerariae Radix Extracts on Antioxidant Enzymes in Ethanol-Treated Rats

The American Journal of Chinese Medicine ◽

10.1142/s0192415x01000368 ◽

2001 ◽

Vol 29 (02) ◽

pp. 343-354 ◽

Cited By ~ 18

Author(s):

Mi-Kyung Lee ◽

Soo-Yuel Cho ◽

Joo-Yeun Jang ◽

Myung-Sook Choi ◽

Seon-Min Jeon ◽

...

Keyword(s):

Body Weight ◽

Antioxidant Enzymes ◽

Control Level ◽

Treated Group ◽

Ethanol Administration ◽

Control Group ◽

Mrna Levels ◽

Water Extracts ◽

Puerariae Radix ◽

Puerariae Flos

This study was performed to investigate the effect of Puerariae Flos (PF) and Puerariae Radix (PR) water extracts on the activities and mRNA expression of three hepatic antioxidant enzymes in ethanol-treated rates. Male Sprague-Dawley rats were divided into four groups, a control, ethanol-treated, ethanol plus PF-treated, and ethanol plus PR-treated group with seven rats per group. Ethanol (25% v/v, 5g/kg body weight) was orally administered once a day for 5 weeks. The PF and PR water extracts were supplemented in a diet based on 1.2 g of raw PF or PR/kg body weight/day. Ethanol administration without the PF or PR supplement significantly lowered the activities of hepatic Cu/Zn SOD and catalase (CAT), whereas it increased the hepatic GSH-Px activity. However, the PF and PR supplementation resulted in a significant increase in the Cu/Zn SOD and/or CAT activities and a significant decrease in the GSH-Px activity in the ethanol-treated rats. The mRNA levels of these antioxidant enzymes in the ethanol-treated rats were normalized to the control level by the PF or PR supplement. The hepatic glutathione content, which was significantly lower in the ethanol-treated group than in the control group, was also normalized to the control level by supplementing with either PF or PR. The PF or PR supplement resulted in lowering the hepatic malondialdehyde to the control level in the ethanol-treated rats.

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Lymphedema alters lipolytic, lipogenic, immune and angiogenic properties of adipose tissue: a hypothesis-generating study in breast cancer survivors

Scientific Reports ◽

10.1038/s41598-021-87494-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Michal Koc ◽

Martin Wald ◽

Zuzana Varaliová ◽

Barbora Ondrůjová ◽

Terezie Čížková ◽

...

Keyword(s):

Breast Cancer ◽

Adipose Tissue ◽

Cancer Survivors ◽

Breast Cancer Survivors ◽

Complex Analysis ◽

Control Group ◽

Mrna Levels ◽

Unilateral Breast Cancer ◽

Cell Content

AbstractLater stages of secondary lymphedema are associated with the massive deposition of adipose tissue (AT). The factors driving lymphedema-associated AT (LAT) expansion in humans remain rather elusive. We hypothesized that LAT expansion could be based on alterations of metabolic, adipogenic, immune and/or angiogenic qualities of AT. AT samples were acquired from upper limbs of 11 women with unilateral breast cancer-related lymphedema and 11 healthy women without lymphedema. Additional control group of 11 female breast cancer survivors without lymphedema was used to assess systemic effects of lymphedema. AT was analysed for adipocyte size, lipolysis, angiogenesis, secretion of cytokines, immune and stem cell content and mRNA gene expression. Further, adipose precursors were isolated and tested for their proliferative and adipogenic capacity. The effect of undrained LAT- derived fluid on adipogenesis was also examined. Lymphedema did not have apparent systemic effect on metabolism and cytokine levels, but it was linked with higher lymphocyte numbers and altered levels of several miRNAs in blood. LAT showed higher basal lipolysis, (lymph)angiogenic capacity and secretion of inflammatory cytokines when compared to healthy AT. LAT contained more activated CD4+ T lymphocytes than healthy AT. mRNA levels of (lymph)angiogenic markers were deregulated in LAT and correlated with markers of lipolysis. In vitro, adipose cells derived from LAT did not differ in their proliferative, adipogenic, lipogenic and lipolytic potential from cells derived from healthy AT. Nevertheless, exposition of preadipocytes to LAT-derived fluid improved their adipogenic conversion when compared with the effect of serum. This study presents results of first complex analysis of LAT from upper limb of breast cancer survivors. Identified LAT alterations indicate a possible link between (lymph)angiogenesis and lipolysis. In addition, our in vitro results imply that AT expansion in lymphedema could be driven partially by exposition of adipose precursors to undrained LAT-derived fluid.

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Identification for antitumor effects of tramadol in a xenograft mouse model using orthotopic breast cancer cells

Scientific Reports ◽

10.1038/s41598-021-01701-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Myoung Hwa Kim ◽

Jeong-Rim Lee ◽

Ki-Joon Kim ◽

Ji Hae Jun ◽

Hye Jeong Hwang ◽

...

Keyword(s):

Breast Cancer ◽

Nk Cell ◽

Cell Activity ◽

Treated Group ◽

Control Group ◽

Nk Cell Activity ◽

Clinical Dose ◽

Group 2 ◽

Mcf 7 ◽

Group 1

AbstractIn our previous research showed that tramadol having potential anti-tumor effect was associated with enhancement of oncological prognosis in patients with breast cancer surgery. As these effects have not been confirmed by clinical dose-regulated animal or prospective human studies, we investigated the anti-tumor effect of tramadol in vivo. Female nude mice orthotopically inoculated with luciferase-expressing MCF-7 cells, were randomly divided into the control (saline), tramadol group 1 (1.5 mg kg−1 day−1), tramadol group 2 (3 mg kg−1 day−1), and morphine (0.5 mg kg−1 day−1) (n = 5/group). Bioluminescence signals after D-luciferin injection, tumor size, and tumor weight were compared among groups after 4 weeks. Estrogen receptor (ER), progesterone receptor (PR), and transient receptor potential vanilloid (TRPV)-1 expression, natural killer (NK) cell activity, and serum interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6 were then examined. Tumour growth was attenuated in tramadol-treated groups (P < 0.05). NK cell activity was significantly decreased only in the morphine treated group not in sham, control, and tramadol groups. The expression levels of ERα, PRα and β, and TRPV1 were decreased in tramadol group 2 compared with those in the morphine group, but not compared to the control group. Serum levels of IL-6 and TNFα were reduced in both tramadol-treated group 1 and 2 compared to the control group. Overall, clinical dose of tramadol has anti-tumour effects on MCF-7 cell-derived breast cancer in a xenograft mouse model.

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