Anticarcinogenic Plants for Oncology: Nutraceuticals Replacing Traditional Chemotherapy? An in-vitro & in-vivo Investigation with Zebrafish models of Human Breast Cancer
Cancer Genetics and Therapeutics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
Despite the advancements in targeted radiation and chemotherapy, these treatments still have limitations and pose risks to healthy tissue. However, alternative therapies, also referred to as nutraceuticals, offer potential solutions in the fight against different diseases including breast cancer without harmful side effects. Screening herbal extracts such as persimmon leaves and Mediterranean herbs like marjoram and thyme is the research avenue to determine their therapeutic indexes and the translational applicability for human patients. Through our observations and phytochemical constituents, these natural remedies have demonstrated a range of beneficial properties, including anticarcinogenic, apoptotic, pro-metabolite, and antioxidant effects.
Methods:
Nutraceutical Preparation: The extraction and preparation of alternative therapeutics was completed using microwave-assisted ethanol extraction (ETHOS-X) and subsequent rotary evaporation, followed by a drying period.
Cell Culturing and Selection: Cell cultures were maintained according to ATCC guidelines with evidence-based modifications for healthy breast MCF-12A and MCF-12F as control lines, hormonal positive BT-483, and MCF7, and triple-negative HTB-26 and HCC-38.
Metabolomics: Biolog plates PMM (Phenotype Mammalian Microarray 1-8) were used to formulate metabolomics profiles in the form of normalized spectra and absorbance readings. This measures the amount of NADH produced in the presence of different molecules such as, but not limited to, metabolic effectors, amino acids, hormones, and ions.
Enrichment, Pathway, and Metabolic Analysis: Used to validate pathway interactions in alternative therapeutics, human cancer cells, and zebrafish biological systems. Metabolism and metabolic analysis are useful to determine which components are causal to therapeutic effect.
MTS assay: Conducted to evaluate cell viability, cytotoxicity, and proliferation after treatment with all 3 alternative therapeutics at set concentrations.
In vivo Zebrafish (Danio rerio) Injections and Treatment: 5D, Tg(fli1:EGFP), zebrafish are raised, bred, and kept according to ZIRC and Clemson IACUC husbandry standards. Six breast cancer cell lines are fluorescently labeled in culture, incubated, prepared, and subsequently injected into the upper region of the yolk sac at 2 days post fertilization. The embryos are treated to remove pigmentation, allowing for visualization and quantification of metastasis, tumor growth, vasculature, and response to treatment. Treatment is via immersion for 6 days.
Gene Expression: Profiles of furthest metastasis sites using head and tail digests. Detection follows RNA extraction, cDNA preparation, and qRT-PCR.
Analysis: Highlights the previously reported cancer-related pathways’ altered regulation (anti-carcinogenic mechanisms) in the presence of phytochemicals (phenolic compounds, terpenes) of interest, such as within the Nf-κB/PI3K/Akt/JNK/MAPK pathway(s) related to cell proliferation, cell-cycle regulation, and apoptosis as seen in-vitro, and connects to metabolic data.
Results:
Key findings include a confirmed impact of selected plant extracts on cancer cells’ energy production, indicated through the NADH produced–and specifically the reduction of anaerobic metabolism. Responses driven by nutraceuticals to certain hormones suggest potential targets for future therapeutic tailoring. Furthermore, no deleterious, cytotoxic effects are observed on healthy cells, unlike doxorubicin. In the zebrafish models, deleterious, lethal effects are observed on xenograft models from immersion in doxorubicin, unlike the nutraceutical therapeutics, with a proposed anti-carcinogenic effect within the therapeutic range.
Conclusion:
Through our observations, these nutraceuticals have demonstrated a range of beneficial properties, including anticarcinogenic, apoptotic, and pro-metabolite effects. This indicates a high potential for their use in treatment protocols. This also serves as confirmatory research of their mechanism(s) of action. With the indicated high potential for the use of natural compounds in treatment protocols for breast cancer, future studies on mammalian in-vivo models and human subjects are necessary to validate and expand these results.
Despite the advancements in targeted radiation and chemotherapy, these treatments still have limitations and pose risks to healthy tissue. However, alternative therapies, also referred to as nutraceuticals, offer potential solutions in the fight against different diseases including breast cancer without harmful side effects. Screening herbal extracts such as persimmon leaves and Mediterranean herbs like marjoram and thyme is the research avenue to determine their therapeutic indexes and the translational applicability for human patients. Through our observations and phytochemical constituents, these natural remedies have demonstrated a range of beneficial properties, including anticarcinogenic, apoptotic, pro-metabolite, and antioxidant effects.
Methods:
Nutraceutical Preparation: The extraction and preparation of alternative therapeutics was completed using microwave-assisted ethanol extraction (ETHOS-X) and subsequent rotary evaporation, followed by a drying period.
Cell Culturing and Selection: Cell cultures were maintained according to ATCC guidelines with evidence-based modifications for healthy breast MCF-12A and MCF-12F as control lines, hormonal positive BT-483, and MCF7, and triple-negative HTB-26 and HCC-38.
Metabolomics: Biolog plates PMM (Phenotype Mammalian Microarray 1-8) were used to formulate metabolomics profiles in the form of normalized spectra and absorbance readings. This measures the amount of NADH produced in the presence of different molecules such as, but not limited to, metabolic effectors, amino acids, hormones, and ions.
Enrichment, Pathway, and Metabolic Analysis: Used to validate pathway interactions in alternative therapeutics, human cancer cells, and zebrafish biological systems. Metabolism and metabolic analysis are useful to determine which components are causal to therapeutic effect.
MTS assay: Conducted to evaluate cell viability, cytotoxicity, and proliferation after treatment with all 3 alternative therapeutics at set concentrations.
In vivo Zebrafish (Danio rerio) Injections and Treatment: 5D, Tg(fli1:EGFP), zebrafish are raised, bred, and kept according to ZIRC and Clemson IACUC husbandry standards. Six breast cancer cell lines are fluorescently labeled in culture, incubated, prepared, and subsequently injected into the upper region of the yolk sac at 2 days post fertilization. The embryos are treated to remove pigmentation, allowing for visualization and quantification of metastasis, tumor growth, vasculature, and response to treatment. Treatment is via immersion for 6 days.
Gene Expression: Profiles of furthest metastasis sites using head and tail digests. Detection follows RNA extraction, cDNA preparation, and qRT-PCR.
Analysis: Highlights the previously reported cancer-related pathways’ altered regulation (anti-carcinogenic mechanisms) in the presence of phytochemicals (phenolic compounds, terpenes) of interest, such as within the Nf-κB/PI3K/Akt/JNK/MAPK pathway(s) related to cell proliferation, cell-cycle regulation, and apoptosis as seen in-vitro, and connects to metabolic data.
Results:
Key findings include a confirmed impact of selected plant extracts on cancer cells’ energy production, indicated through the NADH produced–and specifically the reduction of anaerobic metabolism. Responses driven by nutraceuticals to certain hormones suggest potential targets for future therapeutic tailoring. Furthermore, no deleterious, cytotoxic effects are observed on healthy cells, unlike doxorubicin. In the zebrafish models, deleterious, lethal effects are observed on xenograft models from immersion in doxorubicin, unlike the nutraceutical therapeutics, with a proposed anti-carcinogenic effect within the therapeutic range.
Conclusion:
Through our observations, these nutraceuticals have demonstrated a range of beneficial properties, including anticarcinogenic, apoptotic, and pro-metabolite effects. This indicates a high potential for their use in treatment protocols. This also serves as confirmatory research of their mechanism(s) of action. With the indicated high potential for the use of natural compounds in treatment protocols for breast cancer, future studies on mammalian in-vivo models and human subjects are necessary to validate and expand these results.