OSA: Herbal Management of Canine OSA Patients

This exclusive article is part of the Veterinary Professional Blog Series

This article is the property of Dr. Erin Bannink, December 2020.

Do not distribute without written permission.

IN THIS ARTICLE

I am not accepting any more Teleconsultation requests at this time.

INTRODUCTION

Osteosarcoma is the most common pediatric primary bone tumor worldwide and is associated with a high metastatic rate and poor prognosis. Initially, survivals were marginally improved over surgery alone with the introduction of chemotherapy. But survivals have not further improved in recent decades. Overall survival rate in the human population is only 30%. New therapies for this disease are desperately needed.

Canine Osteosarcoma shares many common molecular pathways with human osteosarcoma and is an attractive model for comparative research. Because osteosarcoma occurs approximately 10 times more frequently in dogs than in people and the survivals in our canine patients are equally dismal, exploration of novel therapies to improve survivals becomes an interesting prospect not only for our canine patients but for its potential implications in translational research.

Numerous signaling pathways important in the progression of OSA have been identified including Notch, Wnt, hedgehog, PI3K/AKT-mTOR, Ras-Raf-MEK-ERK, Ezrin, and JAK/STAT signaling pathways. These pathways are inhibited by many natural compounds including curcumin, resveratrol, apigenin (found in many fruits and vegetables), and sulforaphanes (found in cruciferous vegetables). Findings such as these suggest not only that the foods and supplements we are ingesting might play a role in disease control, but also that herbal medicines, which also contain many compounds proven to impact these tumor progression pathways may play an interesting and promising role in long term management of osteosarcoma. For example, Berberine which is found in a number of Traditional Chinese Herbal Medicinal plants like Scutellaria, Bupleurum, Rhubarb and Coptis, induces apoptosis in human OSA cells through inhibition of PI3K/Akt signaling.

In this month's article I will share outcomes seen in my practice using herbal therapies after amputation when chemotherapy was declined due to cost or client preference. We will then cover some potential mechanisms of action which may explain the outcomes I am seeing in these patients.

SURVIVAL STATISTICS

Following are preliminary survival statistics for patients I have treated in my practice with various integrative protocols and informed consent after amputation. Patient data is still maturing. I see many clients specifically for herbal management in patients for whom chemotherapy has already been declined. For your information I have also included a general summary of published statistics with conventional interventions. (MST = median survival time)

While large studies which are appropriately powered are required to define statistically significant results, the below patient survivals suggest there may be promise for comprehensive herbal intervention as alternative or complementary treatment option for patients with this disease. My practice is seeing survival times in patients treated with amputation and herbal therapies that rival those of amputation and chemotherapy. Additionally, survival in patients treated with an integrative approach combining Chinese Herbal therapies and chemotherapy shows survival times that do not appear to be worse than chemotherapy alone, indicating a low likelihood that Chinese Medicine herbal therapies are decreasing efficacy of chemotherapy treatments in my patients. This clinical experience is sound rationale for supporting further research into the potential role of herbal therapies in management of dogs with appendicular osteosarcoma after amputation. Immunotherapies are gaining wide attention for this disease and it is possible that herbal therapies may act in similar ways to improve survival after amputation.

Amputation alone:

Amputation and Chemotherapy:

Stereotactic radiation and chemotherapy:

Amputation and herbal therapies without chemotherapy (8 patients):

Amputation, chemotherapy and herbal therapies (7 patients)

THE POTENTIAL ROLE OF CHINESE HERBAL THERAPIES IN OSA MANAGEMENT

Epidermal Growth Factor Receptor and OSA progression

Epidermal growth factor receptor (EGFR) is important in the metastatic behavior of osteosarcoma and has been identified as a potential therapeutic target given its role in OSA cell proliferation and frequent over-expression in canine OSA tumor tissue. High EGFR expression, seen in 75% of canine pulmonary metastasis samples studied, was associated with shorter survival time and disease free interval in one study. This high EGFR expression was also seen in the primary tumor tissue in these cases.

Huang Qin | Scutellaria | Wogonin, Berberine

Huang Lian | Coptis | Berberine

Da Huang | Rhubarb | Berberine

Chai Hu | Bupleurum | Berberine

Wu Wei Zi | Schisandra | Gomisin N

Wogonin, isolated from Scutellaria (Huang Qin), dose-dependently inhibited the growth of tumor cells which expressed high levels of EGFR. Gomisin N, isolated from Schisandra, inhibits activation of EGFR, as well as suppresses NF-kB signaling, both of which enhance TNF-alpha induced apoptosis. Berberine, found in a number of TCM herbs (Scutellaria, Coptis, Rhubarb, Bupleurum), suppressed growth of prostate cancer cells in vitro via inhibition of EGFR signaling, leading to apoptosis and cell cycle arrest.

As a maintenance therapy after chemotherapy herbs may work by enhancing immune surveillance for cancer cells and also by antiangiogenic mechanisms, inhibiting the blood supply to newly forming cancer masses which is a similar mechanism to metronomic chemotherapy protocols that accomplish this with pharmaceuticals. There may be additional mechanisms by which herbs provide beneficial effects in osteosarcoma patients.

Alternative or Integrative Curative Intent Therapy: The OVRS Experience

Prolonged survivals have been regularly seen in my practice with herbal medicine therapy alone after amputation or as part of an integrative approach with chemotherapy. Most patients receive vitamin D3 supplementation, monthly acupuncture, and at least two TCM herbal formulas at high doses along with counseling about providing a nutritionally balanced fresh food diet. Herbal medications are prescribed based on TCM prescribing principles. Pharmaceutical grade sustained release curcumin, high dose medicinal mushrooms, probiotics and digestive enzymes are also often included in the treatment protocol. This protocol is instituted as a lifestyle change for the patient, intended to be continued for life. Adjustments are made in the herbal formulas as appropriate based on exam findings and history during rechecks.

The most commonly used formulas for OSA management in my practice are:

1) OSA study formula (see previous article): especially for patients with Toxic Heat signs

2) Xiao Chai Hu Tang (or some modification)

3) Xue Fu Zhu Yu Tang modified with E Zhu and San Leng, often with the addition of a formula called Bao Hu Jian Jun Tang sold by Natural Path which is a Liver Blood tonic

4) Bu Zhong Yi Qi Tang: most commonly needed for patients also receiving chemotherapy

5) Jiu Jun Zi Tang: especially in patients receiving chemotherapy who need appetite support

6) Gui Pi Tang: most commonly needed for patient who need bone marrow support, particularly of platelet counts, during chemotherapy

Below I will describe some of the research behind mechanisms of action for Xiao Chai Hu Tang, one of the most common herbal formulas I prescribe in management of osteosarcoma.

XIAO CHAI HU TANG AND OSTEOSARCOMA

Chai Hu | Bupleurum | Saikosaponins A, C and D

Huang Qin | Scutellaria | Baicalein, Wogonin

Ren Shen | Ginseng | Ginsenosides Rg1, Rh2, Rb3, Rg3, and Rf

Sheng Jiang | Ginger | 6-gingerol

Gan Cao | Licorice | Licochalcone A, Glabridin

Zhi Ban Xia | Pinellia |

Da Zao | Jujube |

Prescribing Xiao Chai Hu Tang

Above are the ingredients for the Classical Chinese Herbal Formula Xiao Chai Hu Tang, or Minor Bupleurum, and their major constituents studied for anticancer activity. Xiao Chai Hu Tang is one of the most widely used herbal formulas in the world given its widespread use in treating viral disease, respiratory infections, various hepatopathies and cancer. As such, this formula is commonly produced in versions which replace the Ren Shen (Panax Ginseng) with less expensive herbs. This makes those formulations less suitable for use as an anticancer agent in most patients. Those modified versions are sold by the same name so if you are prescribing this formula make sure to check the ingredients for the product you are using. I use the Natural Path or Evergreen Versions in my practice.

Xiao Chai Hu Tang is indicated for treatment of a condition called "Shao Yang Syndrome" in Chinese Medicine. I typically prescribe this formula for patients who have a wiry pulse and/or who have notable improvement in pulse quality with needling or acupressure on gall bladder points like GB 34, GB 25, BL19. I find the tongue, based again on Chinese Medical assessment, of patients who can benefit from this formula to be highly variable and will often modify the formula with additional herbs depending on patient presentation. In general, if I have a patient with a very "deficient" or weak pulse I usually avoid this formula.

Monitoring and Potential Toxicities

While toxicities have been reported for humans taking this formula, the incidence is low in comparison to the number of people consuming this formula around the world. Liver and pulmonary toxicities are the most frequently cited concerns, and have been most widely reported in Japan and China where the formula is widely available. I have not seen this as a clinically relevant problem in my patients, however I prescribe this formula based on classical Chinese Medicine prescribing principles and follow, minimally, the guidelines I outline above. I also regularly monitor chemistry panels on all my patients receiving chronic herbal therapies and am very particular about the brands and formulations that I prescribe.

In at least one article reviewing reported toxicities with this formula, the authors state, "We revisited the [Xiao Chai Hu Decoction toxicity] event which occurred in late 1980s in Japan and the Bupleuri Radix related adverse drug reaction reports in China. After careful review, comparison, analysis and evaluation, we think the interstitial pneumonitis, drug induced Liver injury and other severe adverse drug events including death [which] happened in Japan [are] probably results from multiple factors, including combinatory use of Xiao Chai Hu Tang with interferon, Kampo usage under modern medicine theory guidance, and use of Xiao Chai Hu Tang on the basis of disease diagnosis instead of traditional Chinese syndrome complex differentiation." (Wu, 2014)

In another recent review evaluating toxicities with this formula and their causes in humans, cases of hepatotoxicity were generally mild to moderate in severity and rapidly reversible with discontinuation of the formula. Recurrence of toxicity after restarting the herbal formula in affected patients was common and is not advised. The mechanism of liver toxicity is not well defined, but may be related to its effects on MMP-9 and caspase-3. It is possible that many of the toxicities could be attributable to contaminants or misidentification of herbal ingredients, which is an unfortunately common situation in formulas manufactured and sold from companies or countries with limited regulation. This is one of the reasons I strongly advise against clients purchasing Chinese herbal formulas over the internet.

Of significant importance for us to consider in veterinary practice is the lack of documented toxicity in multiple toxicity studies in rats. Xiao Chai Hu Tang given orally at a dose of 2g/kg/day for 13 weeks in rats showed no toxicity. There were no liver or kidney alterations on histopathology and no toxicological changes in mortality, weight, water or food consumption, UA, CBC, blood chemistry values, ophthalmic exam, organ weights or clinical signs. Another study also corroborated the no-observed-adverse-effect dose of 2g/kg/day when given for by mouth for 4 weeks. In clinical practice I would typically prescribe 0.5g/25lb BID which amounts to about 0.1g/kg/day, well below the doses used in these studies.

[Herb drug interaction considerations will be addressed in another article, so you don't have to process that all now. You're welcome :) ]

Potential Mechanisms of Anticancer Action

There are many laboratory research articles and preclinical studies published on Xiao Chai Hu Tang as a whole formula. A quick search on pubmed returns 360 articles on the whole formula. Of particular interest to the topic of its use as a support of patients with cancer are the many articles outlining its immune-modulatory effects. That is too large a topic to tackle in this already lengthy article, so will be explored later. But it bears mentioning here that there is possibly much to its action as a potential immunotherapy for osteosarcoma patients, which may explain some of the promising survivals I am seeing in clinical practice when used as a palliative intervention for patients for whom chemotherapy is declined.

Bupleurum chinensis (Chai Hu)

The immunomodulatory activity of Bupleurum species has been attributed primarily to saikosaponins, which activate phagocytic activity in macrophages, suppress T cell response and increase B cell responses. These effects were largely related to down-regulation of NF-kB signaling which decreases the production of pro-inflammatory cytokines. Its anti-inflammatory action is closely associated with its immunomodulating activity, with saikosaponins (A, C and D) primarily involved in its effects to reduce both acute and chronic inflammatory responses. The anti-inflammatory effects were shown to be observable with oral administration in mouse models.

Compounds found in Bupleurum also show anti-tumor activity in various cancer cell lines including leukemia, melanoma, hepatocellular carcinoma, gastric adenocarcinoma and cervical cancer. Specific to osteosarcoma, Saikosaponin D inhibited proliferation of OSA cell lines, caused cell cycle arrest and induced apoptosis at concentrations of 80 µmol/L. Saikosaponin D was shown in another study to significantly inhibit proliferation of osteosarcooma cells as well as limit their invasion and migration. Cytochrome C release was enhanced by the compound. An increased Bax/Bcl-2 ratio and activation of caspase-3, -8 and -9 were observed, indicating that cellular apoptosis was induced by way of both the mitochondrial and death receptor pathways. The study proposed that Saikosaponin D could be investigated as a potential treatment for osteosarcoma based on the theoretical basis for efficacy supported by these in vitro results.

Scutellaria baicalensis (Huang Qin)

Scutellaria is traditionally used in TCM for its anti-inflammatory and antineoplastic action. Baicalein, a flavone found in the roots of Scutellaria, was found to effectively induce apoptosis in three canine OSA cell lines and did not interfere with doxorubicin’s anti-proliferative action. In vivo, baicalin retarded growth of human OSA tumors in mice.

Baicalein has exhibited the ability to inhibit the growth, migration, and invasion of OSA cell lines and induce apoptosis of OSA cell lines via numerous pathways including Ezrin, AKT, and TGF- β1 pathways. The compound also generated reactive oxygen species (ROS) in human OSA cell lines, and induced cell apoptosis via suppression of Bcl-2 and activation of caspase-9 and caspase-3.

Wogonin, isolated from Scutellaria, reduced tumor growth and incidence of metastasis, angiogenesis, lymphangiogenesis and the number of Tumor Associated Macrophages (TAM) in vivo in osteosarcoma-bearing mice models. It has shown similar anti-neoplastic and anti-angiogenic activity in other cancer cell lines. Wogonin also demonstrated the ability to increase Reactive Oxygen Species (ROS) in human OSA cells and altering mitochondrial membrane potential in vitro. These effects were time- and dose-dependent. Induction of apoptosis was observed, via increase in activity of Caspase-3. Increase in other pro-apoptotic proteins (Bad, Bax), and decrease levels of anti-apoptotic proteins (Bcl-2) was also observed. Anti-inflammatory action in human OSA cell lines expressing COX-2 was observed with a Wogonin extract in vitro. The extract also decreased edema in vivo in a mouse model of experimentally induced intra-articular swelling.

Panax Ginseng (Ren Shen)

Ginsenosides from Panax Ginseng have been shown in hundreds of studies to exert anti-neoplastic action on various cell lines via numerous pathways. A few studies have been published evaluating their anti-neoplastic action against OSA specifically. Ginsenoside Rb3, one of the main active constituents in Panax Ginseng, showed dose-dependent cytotoxic action against four human OSA cell lines in vitro. Rg3 has been shown to inhibit the proliferation, migration, and invasion of OSA cells through mechanisms involving downregulation of MMP2, MMP7, and MMP9 expression as well as down-regulation of the Wnt/β-catenin pathway.

Ginsenoside Rf induced cell cycle arrest and apoptosis, via activation of caspase-3 and caspase-9, in a dose-dependent manner in human OSA cell lines.

The ginsenoside Rg1 was shown to act synergistically with Timosaponin AIII (TA3), found in Anamarrhena (The Chinese herb Zhi Mu), to suppress key regulators in cancer metastasis: JNK, p38, ERK, β-catenin, and CREB signaling. These effects were specifically documented in osteosarcoma cell lines in a study investigating potential new drug development for osteosarcoma.

The ginsenoside Rh2 was evaluated for anticancer activity in osteosarcoma cells and shown to exert anticancer effects. Rh2 reduced Bcl-2, caspase 3, and caspase 9 levels, and promote the expression of Bax (a cell death signal). Rh2 also triggered apoptosis by promoting the MAPK signaling pathway and inhibiting PI3K/Akt/mTOR and nuclear factor-κB (NF-κB) signaling pathways.

Interestingly, despite the widespread consensus that herbs should not be administered concurrently with radiation therapy due to likelihood of decreasing the cell killing effect of radiation, ginsenosides have been studied for their potential radiation sensitizing effects. Ginseng polysaccharides have been shown inhibit proliferation and increase apoptosis and autophagy in osteosarcoma cells treated with radiation, leading to the conclusion they could be considered as a potential auxiliary drug to improve radiation cell kill in osteosarcoma patients.

Panax ginseng is a common ingredient in many TCM formulas. Formulas which include Panax Ginseng which are commonly used in my practice in management of cancer include: Bu Zhong Yi Qi Tang, Chai Hu Jia Long Gu Mu Li Tang, Gui Pi Tang, and Xiao Chai Hu Tang.

Ginger (Jiang Sheng)

The anti-cancer effects of 6-gingerol on osteosarcoma cells were investigated and found to be related to activation of caspase cascades and regulation of Bcl2 and Bax. 6-gingerol also activated AMP-activated protein kinase (AMPK) signaling associated with the apoptotic pathways. THese findings suggest potential anticancer action in osteosarcoma cell lines attributable to activation of cell death signaling and the inhibition of cell-survival signaling.

Licorice (Gan Cao)

Licochalcone A is a flavonoid extracted from licorice root. It has been shown to exert anti-proliferative effects on osteosarcoma cells through activation of ATM-Chk2 checkpoint pathway and has been proposed as a potential anti-osteosarcoma therapeutic agent. Another study showed in vitro and in vivo anti-tumor action of Licochalcone against human osteosarcoma cells involving p38MAPK regulation of mitochondria-mediated intrinsic apoptotic pathways.

Another compound found in licorice root, Glabridin, has shown ability to impact anti-metastatic mechanisms in osteosarcoma both in vitro and in vivo. This occurred via multiple pathways including reduction of MMP-2 and MMP-9 expression, G2/M cell cycle arrest, and interference with CREB and AP1 DNA binding.

CONCLUSION

In my clinical experience, after amputation, it appears to be possible to achieve survival times with Traditional Chinese Medicine similar to, or exceeding, those achievable with chemotherapy. Large scale clinical trials would be needed to evaluate the true repeatability and response rates seen with these alternative medicine treatments. However, numerous individual case examples of prolonged survival should raise interest in the role of herbal therapies in management of patients with appendicular osteosarcoma and are the first step in supporting a more extensive evaluation of the role of these interventions in management of this aggressive disease.

Novel approaches are necessary if we are to achieve longer disease remissions in this aggressive disease. Current chemotherapy recommendations at my practice involve single agent Carboplatin therapy with or without metronomic chemotherapy and/or herbal medicine treatments for maintenance therapy. Consultations always include a thorough discussion of the pros and cons of treatments and the current published information about these therapies to support the client in making an informed decision.

REFERENCES:

Introduction: Conventional Treatment and Translational Research

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Kreilmeier T, Sampl S, Deloria AJ, et al. Alternative lengthening of telomeres does exist in various canine sarcomas. Mol Carcinog. 2017;56(3):923-935.

Gatti M, Wurth R, Vito G, et al. Canine osteosarcoma cell lines contain stem-like cancer cells: biological and pharmacological characterization. Jpn J Vet Res. 2016;64(2):101-12.

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Fowles JS, Brown KC, Hess AM, et al. Intra- and interspecies gene expression models for predicting drug response in canine osteosarcoma. BMC Bioinformatics. 2016;17:93.

Shahi MH, York D, Gandour-Edwards R, et al. BMI1 is expressed in canine osteosarcoma and contributes to cell growth and chemotherapy resistance. PLoS One. 2015;10(6):e0131006.

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Selmic LE, Burton JH, Thamm DH, et al. Comparison of carboplatin and doxorubicin-based chemotherapy protocols in 470 dogs after amputation for treatment of appendicular osteosarcoma. J Vet Intern Med. 2014;28(2):554-63.

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Potential Role of Herbal Therapies

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He H1, Han S, Zhang T, et al. Screening active compounds acting on the epidermal growth factor receptor from Radix scutellariae via cell membrane chromatography online coupled with HPLC/MS. J Pharm Biomed Anal. 2012;62:196-202.

Waiwut P1, Shin MS, Inujima A, et al. Gomisin N enhances TNF-α-induced apoptosis via inhibition of the NF-κB and EGFR survival pathways. Mol Cell Biochem. 2011;350(1-2):169-75.

Huang ZH, Zheng HF, Wang WL, et al. Berberine targets epidermal growth factor receptor signaling to suppress prostate cancer proliferation in vitro. Mol Med Rep. 2015;11(3):2125-8.

XIAO CHAI HU TANG

Xiao Chai Hu Tang Toxicity

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Bupleurum (Chai Hu)

Zhao L, Li J, Sun ZB, et al. Saikosaponin D inhibits proliferation of human osteosarcoma cells via the p53 signaling pathway. Exp Ther Med. 2019;17(1):488–494.

Gao T, Zhao P, Yu X, et al. Use of Saikosaponin D and JNK inhibitor SP600125, alone or in combination, inhibits malignant properties of human osteosarcoma U2 cells. Am J Transl Res. 2019;11(4):2070–2080. Published 2019 Apr 15.

Yu JQ, Deng AJ, Wu LQ, et al. Osteoclast-inhibiting saikosaponin derivatives from Bupleurum chinense. Fitoterapia. 2013;85:101-8.

Zhou C, Liu W, He W, et al. Saikosaponin a inhibits RANKL-induced osteoclastogenesis by suppressing NF-κB and MAPK pathways. Int Immunopharmacol. 2015;25(1):49–54.

Shin JE, Kim HJ, Kim KR, et al. Type I saikosaponins a and d inhibit osteoclastogenesis in bone marrow-derived macrophages and osteolytic activity of metastatic breast cancer cells. Evid Based Complement Alternat Med. 2015;2015:582437.

Gao T, Zhao P, Yu X, et al. Use of Saikosaponin D and JNK inhibitor SP600125, alone or in combination, inhibits malignant properties of human osteosarcoma U2 cells. Am J Transl Res. 2019 Apr 15;11(4):2070-2080.

Scutellaria (Huang Qin)

Helmerick EC, Loftus JP, Wakshlag JJ. The effects of baicalein on canine osteosarcoma cell proliferation and death. Vet Comp Oncol. 2014 Dec;12(4):299-309.

Liu Y, Hong Z, Chen P, et al. Baicalin inhibits growth and induces apoptosis of human osteosarcoma cells by suppressing the AKT pathway. Oncol Lett. 2019;18(3):3188–3194.

Wang Y, Wang H, Zhou R, et al. Baicalin Inhibits Human Osteosarcoma Cells Invasion, Metastasis, and Anoikis Resistance by Suppressing the Transforming Growth factor-β1-induced Epithelial-To-Mesenchymal Transition. Anticancer Drugs. 2017;28(6), 581-587.

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Wan D, Ouyang H. Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway. Nat Prod Res. 2018;32(16),1996-2000.

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Lin CC, Kuo CL, Lee MH, et al. Wogonin triggers apoptosis in human osteosarcoma U-2 OS cells through the endoplasmic reticulum stress, mitochondrial dysfunction and caspase-3-dependent signaling pathways. Int J Oncol. 2011 Jul;39(1):217-24.

Chang IC, Huang YJ, Chiang TI, et al. Shikonin induces apoptosis through reactive oxygen species/extracellular signal-regulated kinase pathway in osteosarcoma cells. Biol Pharm Bull. 2010;33(5):816-24.

Ginseng (Ren Shen)

Mao X, Jin Y, Feng T, et al. Ginsenoside Rg3 Inhibits the Growth of Osteosarcoma and Attenuates Metastasis through the Wnt/β-Catenin and EMT Signaling Pathway. Evid Based Complement Alternat Med. 2020 Jul 11;2020:6065124.

Lee SY. Ginsenoside Rg1 Drives Stimulations of Timosaponin AIII-Induced Anticancer Effects in Human Osteosarcoma Cells. Evid Based Complement Alternat Med. 2020 Jul 22;2020:8980124.

Li C, Gao H, Feng X, et al. Ginsenoside Rh2 impedes proliferation and migration and induces apoptosis by regulating NF-κB, MAPK, and PI3K/Akt/mTOR signaling pathways in osteosarcoma cells. J Biochem Mol Toxicol. 2020 Aug 6:e22597.

Zhang XY, Sun K, Zhu Q, et al. Ginseng polysaccharide serves as a potential radiosensitizer through inducing apoptosis and autophagy in the treatment of osteosarcoma. Kaohsiung J Med Sci. 2017 Nov;33(11):535-542.

Ginger (Sheng Jiang)

Fan J, Yang X, Bi Z. 6-Gingerol inhibits osteosarcoma cell proliferation through apoptosis and AMPK activation. Tumour Biol. 2015 Feb;36(2):1135-41.

Licorice (Gan Cao)

Shen TS, Hsu YK, Huang YF, et al. Licochalcone A Suppresses the Proliferation of Osteosarcoma Cells through Autophagy and ATM-Chk2 Activation. Molecules. 2019 Jul 2;24(13):2435.

Lin RC, Yang SF, Chiou HL, et al. Licochalcone A-Induced Apoptosis Through the Activation of p38MAPK Pathway Mediated Mitochondrial Pathways of Apoptosis in Human Osteosarcoma Cells In Vitro and In Vivo. Cells. 2019 Nov 14;8(11):1441.

Jie Z, Xie Z, Zhao X, et al. Glabridin inhibits osteosarcoma migration and invasion via blocking the p38- and JNK-mediated CREB-AP1 complexes formation. J Cell Physiol. 2019 Apr;234(4):4167-4178.