Traditional Tibetan Medicine in Cancer Therapy by Targeting Apoptosis Pathways

Cancer is a leading cause of death around the world. Apoptosis, one of the pathways of programmed cell death, is a promising target for cancer therapy. Traditional Tibetan medicine (TTM) has been used by Tibetan people for thousands of years, and many TTMs have been proven to be effective in the treatment of cancer. This paper summarized the medicinal plants with anticancer activity in the Tibetan traditional system of medicine by searching for Tibetan medicine monographs and drug standards and reviewing modern research literatures. Forty species were found to be effective in treating cancer. More importantly, some TTMs (e.g., Ophiocordyceps sinensis, Phyllanthus emblica L. and Rhodiola kirilowii (Regel) Maxim.) and their active ingredients (e.g., cordycepin, salidroside, and gallic acid) have been reported to possess anticancer activity by targeting some apoptosis pathways in cancer, such as Bcl-2/Bax, caspases, PI3K/Akt, JAK2/STAT3, MAPK, and AMPK. These herbs and natural compounds would be potential drug candidates for the treatment of cancer.


INTRODUCTION
Apoptosis, which is also known as programmed cell death, is beneficial to normal cell development, organ growth, and the dynamic balance of tissues (Rogers and Almenri, 2019). Apoptosis is a normal physiological process that plays an important role in the development and dynamic balance of organisms . Defects in apoptosis occur in most types of cancer, such as lung, female breast, prostate, liver, thyroid, and bladder cancers. A large number of studies have shown that regulating and inducing apoptosis are feasible ways for treating cancer (Hoshyar and Mollaei, 2017;Yoon et al., 2018). In vitro and in vivo experiments have demonstrated that the mechanism of apoptosis encompasses extremely complex processes and involve many biological factors, and failure to induce apoptosis is one of the major obstacles to cancer treatment (Li-Weber, 2013). From a mechanistic perspective, apoptosis can be activated by the intrinsic mitochondrial or extrinsic death receptor apoptotic pathway. The intrinsic mitochondrial apoptotic pathway is activated when cells sense directly or indirectly intracellular or extracellular stimuli, such as DNA damage, reactive oxygen species, hypoxia, and Ca 2+ (Tompkins and Thorburn, 2019). These stimuli ultimately disrupt mitochondrial function by inducing the expression and activation of proapoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL, Bax, and Bak (Hoshyar and Mollaei, 2017). By contrast, stimulated extrinsic death receptors can induce the sequential activation of caspase-3, which cleaves target proteins and leads to apoptosis (Tompkins and Thorburn, 2019). Therefore, the development of anticancer agents with apoptosis pathway-related targets has become an important strategy for cancer treatment.
Natural medicines, including plants, animals, and minerals, are the gifts of nature to humans and play an important role in fighting various diseases. Many anticancer drugs that are commonly used in modern medicine, such as paclitaxel, camptothecin, matrine and vinblastine, are derived directly or indirectly from natural sources. Therefore, new anticancer drugs can be discovered from natural plants. In the course of more than 2,000 years of history, a complete theoretical system has been established for traditional Tibetan medicine (TTM). TTM has played an important role in the prevention and treatment of various diseases, such as "Zhui-nai" (འབྲ ས་ནད།), which is similar to cancer in modern medicine (Bauer-Wu et al., 2014). TTM believes that "Zhui-nai" is caused by external factors invading the body, resulting in the dysfunction of the three "stomach fire". These abnormalities can cause indigestion and increase bad blood, which ultimately lead to the dysfunction of the mei-nian loong (རླ ུ ང་མེ ་ མཉམ།), neng-xiao tripa (མཁྲ ི ས་པ་འཇུ ་བྱ ེ ད།), and baekan ni-mu-xie (བད་ཀན་ མྱ ག་བྱ ེ ད།) (Yutuo, 1983). In TTM, unclean substances in the body and physical weakness are important factors in the development of cancer. Therefore, TTM with tonic, heat-clearing and detoxification functions can be used to treat cancer. In recent years, TTM has received extensive attention worldwide owing to its unique advantages in terms of preventing and treating cancer. TTM can directly inhibit the growth of cancer cells, induce apoptosis, and suppress tumor growth through multi-target pathways (Yadav et al., 2017;Bhardwaj et al., 2018;Tao et al., 2019). In addition, TTM combined with radiotherapy or chemotherapy can significantly reduce adverse reactions and enhance the patient's immunity and quality of life (Liu et al., 2016a;Liu et al., 2016b;Colapietro et al., 2018). Numerous TTM monographs and research papers have documented some natural medicines and prescriptions for cancer treatment. However, no consensus has been reached in most records, resulting in a lack of systematic summarization, induction, and arrangement.
In this study, information on natural Tibetan medicines used in treating cancer was sampled by performing a bibliographic investigation of TTM monographs and drug standards. The names, species, families, and medicinal parts of TTMs with anticancer effect were introduced in detail. These data can provide a good reference for the development and utilization of TTMs. Moreover, recent research progress on some anticancer TTMs and their active ingredients that can induce apoptosis in cancer cells was introduced in detail. These herbs and natural compounds would be potential drug candidates for the treatment of cancer.

METHODS
Some Tibetan medicine monographs and medicinal materials standards, such as "Jing Zhu Materia Medica", "Drug Standards of Tibetan Medicine" and "Chinese Tibetan Medicine", were searched for information on natural Tibetan medicine for cancer treatment. Data collected from these documents included names, species, families, and medicinal parts. The botanical names of plants are mainly derived from references, and verified through the "Flora of China (http://frps.eflora.cn/)" and Medicinal Plant Names Services: Royal Botanic Gardens, Kew databases based on their Chinese names. In addition, in order to obtain the active ingredients and biological/pharmacological effects of the selected species, online Chinese databases (e.g., Wanfang and CNKI) and international databases (e.g., NCBI, Web of Science, and Science Direct) were searched with cancer, apoptosis and/or Latin names as search keywords.

RESULTS AND DISCUSSION
Understanding of Cancer in Traditional Tibetan Medicine and organs are healed and repaired, and the systemic immune system is restored to normal. In TTM, unclean substances in the body are considered important factors in the development of cancer. Therefore, TTMs with tonic, and heat-clearing, detoxifying functions are usually used to treat cancer (Table 1 and Figure 2). TTM prescriptions for cancer treatment are mainly based on the six tastes (i.e., sweet, sour, salty, bitter, astringent, and pungent). These tastes transform sequentially into three gastropyretic phases, which become into three post-digestive taste profiles (sweet, sour, and bitter) (Bauer- Dhondrup et al., 2019). Prescription medicines include medicinal plants, animals, and/or minerals, which are processed by powdering, boiling, concentration, and mixing (Dimaer, 2012). TTM also uses some mineral medicines in the treatment of cancer, such as Margarita, Margaritifera concha, Magnetitum, and Cinnabaris, but their modern research is scarce.

Apoptotic Pathway as a Target of TTM in the Treatment of Cancer
Apoptosis, which is known as programmed cell death, is a widely important mechanism of cell growth inhibition in cancer cells. Therefore, the apoptotic pathway is an important target for cancer treatment (Tompkins and Thorburn, 2019). By collating the literature of Tibetan medicines with anticancer activity, up to now, these anticancer TTMs were found among forty species, such as Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M., Crocus sativus L., Phyllanthus emblica L., Rhodiola species, Mirabilis himalaica (Edgew.) Heimerl, Terminalia chebula Retz. Some TTMs can kill cancer cells by inducing apoptosis. In the following sections, we will focus on introducing the TTMs and their compounds whose therapeutic mechanisms are related to apoptosis (Tables 2 and 3).

TTMs That Alter the Bcl-2/Bax Ratio
The Bcl-2 family has both proapoptotic and surviving members, which play important roles in regulating apoptosis (Rogers and Almenri, 2019). As an antiapoptotic protein, Bcl-2 is mainly distributed in the outer membrane of mitochondria, the inner surface of cell membrane, endoplasmic reticulum and nuclear membrane. On the contrary, Bax is a proapoptotic member of Bcl-2 family. Therefore, the proportional relationship between Bax/Bcl-2 plays a key role in mitochondrial mediated apoptosis . In addition, many TTMs have been found to induce apoptosis by regulating the balance of Bcl-2 family members.
Cancer is recognized to be the result of three factors dysfunction (loong, tripa and baekan), especially under the inverse of the loong. According to the ancient Tibetan medicine classics, TTMs with tonic, clearing heat, and detoxifying functions are used to treat cancer. O. sinensis (Figure 2), which is known as Ya-er-zha-geng-bu (Tibetan: དབྱ ར་རྩ ྭ ་དགུ ན་འབུ །), Dong-chong-xia-cao (Chinese name) or cordyceps (English name), is considered as one of the most valued Tibetan medicines (Qinghai Institute for Drug Control, 1996). In the theory of Traditional Chinese medicine and Tibetan medicine, O. sinensis is pungent flavor and warmnatured, and used for hundreds of years in traditional medicine as a tonic for the bronchitis, phthisis, pneumonia, lung heat, and impotence nocturnal emission (Dimaer, 2012). It's worth noting that O. sinensis has a wide range of pharmacological properties, such as anti-inflammatory, cell cycle disruption, immune enhancement, induction apoptosis, etc. So it is widely used and concerned as an anticancer agent. Previous studies have found that water extract of O. sinensis combined with methotrexate could significantly prolong the survival time of mice inoculated with cancer cell sarcoma and inhibit the metastasis of tumor cells by inducing apoptosis (Nakamura, et al., 2003). An exopolysaccharide fraction from O. sinensis could significantly inhibit the metastasis of B16 melanoma cells and decreased the levels of Bcl-2 in the lungs and livers at a dose concentration of 120 mg/kg . Treatment of A549 lung cancer cells with protein extract of O. sinensis could increase in Bax/Bcl-2 ratio, significantly upregulate mRNA levels of Bax, tumor necrosis factor-a (TNF-a), interleukin-1, and interleukin-12 (Wang Y.X. et al., 2018). Tinospora cordifolia (Willd.) Hook.f. & Thomson (Tibetan: Le-zhe) is one of the most widely used in TTM, has immunomodulatory, antitumor, anti-angiogenesis, and antimetastatic activity in various in vivo models. Aqueous ethanolic extract of T. cordifolia could block C6 glioma cells in G0/G1 phase and G2/M phase, inhibit the expression of G1/S phase specific protein cyclin D1 and antiapoptotic protein Bclxl, and thus produce its antiproliferation and apoptotic inducing effect in concentration range of 250-350 mg/ml (Ali and Dixit, 2013).   Inhibition proliferation (Hao, 2014) Neolin SGC-7901 HepG2 A549 Inhibition proliferation (Hao, 2014) 14-O-acetylneoline SGC-7901 HepG2 A549 Inhibition proliferation (Hao, 2014) Songorine SGC-7901 HepG2 A549 Inhibition proliferation (Hao, 2014) 12-epi-napelline SGC-7901 HepG2 A549 Inhibition proliferation (Hao, 2014) 12-epidehydronapelline SGC-7901 HepG2 A549 Inhibition proliferation (Hao, 2014)    Some active ingredients of TTMs that alter the Bcl-2/Bax ratio have been found and identified. Cordycepin, a 3-deoxyadenosine (Figure 3), is the predominant functional component of the fungus Ophiocordyceps species, has antitumor effects or apoptosis in brain cancer, human oral squamous cancer, thyroid carcinoma cancer, gallbladder cancer, liver cancer, breast cancer, and lung cancer (Wu et al., 2007;Aramwit et al., 2015;Chaicharoenaudomrung et al., 2018). Cordycepin (5.11-15.34 mM) could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. It was demonstrated that cordycepin could decrease the expression levels of Bcl-2 and caspase-3, increase the expression levels of proapoptotic protein Bax, and cleaved caspase-3 (Wang Z. et al., 2016). Notably, the study showed that cordycepin (125-500 mM) could induce the mitochondria mediated apoptosis signal pathway of human liver cancer HepG2 cells through upregulation of the ratio of Bax/Bcl-2, and initiating the FADD mediated signal pathway (Shao et al., 2016). Mirabijalone E (Figure 3), which was isolated from M. himalaica, has been reported to increase of Bax expression level and decrease of Bcl-2 level and activation of caspase-3 (Linghu et al., 2014). In addition, chebulagic acid ( Figure 3) which was isolated from the fruits of T. chebula, could induce apoptosis by DNA fragmentation assay, PARP cleavage, cytochrome c release from the mitochondria and alteration of Bcl-2/Bax ratios in COLO-205 cell line (with an IC 50 of 25mM) (Reddy et al., 2009).

TTMs That Activate Caspases
Caspases are a family of cysteine proteases and play an important role in apoptotic and inflammatory signaling pathways. During the process of tumorigenesis, significant loss or inactivation of major members of the caspase family leading to impaired apoptosis induction, causing the serious imbalance of growth dynamics, and eventually to abnormal growth of human tumors (Rogers and Almenri, 2019). Caspases are divided into promoter groups (caspase-8/9/10) and executive groups (caspase-3/6/7). Reactivation of caspase to restore the apoptosis-induced pathway is a key molecular approach to the development of anticancer agents. Restoring apoptosis induction by caspase reactivation is a key molecular mechanism for the development of anticancer agents. Most studies have found that TTMs could induce apoptosis via caspase activation.
Saffron ( Figure 2) is an edible spice and colorant found in the dried stigmas of Crocus sativus L. and has been used in TTM as an herb to treat various diseases, such as cancer. Over the past two decades, studies have been conducted on the therapeutic applications of saffron, which have been found to have anticancer, antitumor (in vivo and in vitro) and antimutagenic potential. Modern pharmacological studies have been proved that saffron can treat a variety of cancers, such as lung, breast, skin, and prostate cancers. In human lung cancer (A549 and H446), saffron extract (0.25-8.0 mg/ml) could suppress proliferation and induce apoptosis in a dose-and time-dependent manner and has significant anticancer effects via caspase-3/8/9 mediated cell apoptosis (Liu et al., 2014). The crocin family includes various glycosyl esters of which six types have been detected in saffron and is the main active substance of saffron. Previous studies have shown that crocin (0.2-1.0 mmol/L) could induce ovarian cancer HO-8910 cells' apoptosis by increasing p53 and Fas/APO-1 expression and activating the apoptotic pathway regulated by Caspase-3 (Xia, 2015). In addition, crocin could induce apoptosis on human breast cancer cells (MCF-7) through a caspase-8-dependent mitochondrial pathway, involving p53 expression, Bax conformation, and mitochondrial membrane potential loss .
Dracocephalum tanguticum Maxim. (Labiatae) is a commonly used TTM for treating arthritis, hepatitis, and ulcer. In recent years, D. tanguticum has been used to treat glioblastomas. Wang et al. (2011) found that the chloroform extract of D. tanguticum Inhibition proliferation (Saleem et al., 2002) Ellagic acid MCF-7 S115 HOS-1 PC-3 PNT1A Inhibition proliferation (Saleem et al., 2002) 40  . The ethanol extract of Stellera chamaejasme L. induced apoptosis significantly improved the activity of caspase-3/8/9, increased Fas and TNF-a expression (Liu X.N et al., 2012). Carum carvi L. essential oil has an efficient novel apoptosis inducer for human colon cancer cells (HT-29 and Huvec) by upregulation Caspase-3 gene expression (Khatamian et al., 2019). Ellagic acid (Figure 3), an important small molecular compound, was isolated from some TTMs, such as P. emblica, T. chebula, and T. billerica. Similarly, ellagic acid is a polyphenolic compound found in fruits and berries such as pomegranate, strawberry, raspberry, and blackberry. A large number of studies have reported the anticancer effects of ellagic acid on most types of cancer, such as colorectal, breast, prostate, lung, and liver cancers (Ceci et al., 2018). Hagiwara et al. (2010) found that ellagic acid activated apoptosis pathway associated with caspase-3 activation in human leukemia HL-60 cells. Notably, ellagic acid could enhance the chemotherapeutic sensitivity of 5-Fluorouracil and induce apoptosis by regulating the Bcl-2/Bax ratios and activating caspase-3 in colorectal carcinoma cells (HT-29) (Kao et al., 2012). In addition to the mechanisms mentioned above, ellagic acid induced apoptosis by regulating ROS, PI3K/Akt, JAK2/STAT3, MAPK, and NF-kB pathway in cancer cells (Bisen et al., 2012;Ceci et al., 2018).

TTMs That Activate Reactive Oxygen Species
Reactive oxygen species (ROS) are substances produced by all aerobic cells to regulate cell development, growth, survival and death. ROS are generally present in all aerobic cells in relative balance with biochemical antioxidants. When this balance is disrupted by mitochondria excess production of ROS and/or depletion of antioxidants, oxidative stress may occur, which eventually leads to mitochondrial swelling, depolarization of mitochondrial membrane potential, and release of apoptosisinducing proteins (Wang R. et al., 2016;Chaicharoenaudomrung et al., 2018). Oxidative stress is a major apoptotic stimulus for cancer cells, which require particularly high energy metabolism in the process of rapid growth and proliferation. Therefore, the production of ROS may enhance the proapoptotic mechanism of cancer cells and provide important targets for the treatment of cancer. TTMs have been reported to induce apoptosis of cancer cells by production of ROS. P. emblica, a euphorbiaceous plant, is widely distributed in subtropical and tropical regions of China, India, Indonesia, and Malay Peninsula (Liu X.L. et al., 2012). The dried fruits of P. emblica is one of the famous plants used in traditional medicinal systems such as Ayurvedic medicine, Tibetan traditional medicine, Chinese herbal medicine, and Thai traditional medicine (Zhang Y.J. et al., 2004;Ngamkitidechakul et al., 2010). In traditional medicine Tibetan system, P. emblica is called "Ju-ru-re" (Tibetan: སྐ ྱ ུ ་རུ ་ར།). It is the most frequently used formulations in TTM (Li et al., 2018a). The extensive use of P. emblica in traditional medicines and food products has led to a large number of pharmacological activity studies. Up to now, a large number of biological activities have been reported, such as anti-inflammatory, antioxidant, antitumor, and immunomodulatory effects. It is noted that the aqueous extract of P. emblica (25-100 mg/ml) could induce apoptosis on human hepatoma cells (HepG2) by reducing production of ROS and increasing the levels of glutathione (Shivananjappa and Josi, 2012).
Swertia mussotii Franch., which is known as "Di-da" (Tibetan: ཏི ག་ཏ།), was reported in the classic book of Tibetan medicine "Jing Zhu Materia Medica" that S. mussotii has the clearing heat and detoxifying functions. Recent studies have shown that S. mussotii has significant anticancer activity.  reported that ethanol extract of S. mussotii was able to induce apoptosis in gastric cancer cells (MGC-803 and BGC-823) through depolymerization of cytoskeletal filaments, S phase arrest, disrupted mitochondrial transmembrane potential and increased cytoplasmic levels of ROS. Similarly, Meconopsis horridula Hook. f. & Thomson ethanol extract induced murine leukemia L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process (Fan et al., 2015b). Gallic acid, 3,4,5-trihydroxybenzoic acid (Figure 3), which can be found in various natural products, such as green tea, grapes, Punica granatum L., P. emblica, Galla chinensis Mill., and many other fruits plants. Gallic acid known to affect several pharmacological and biochemical pathways have strong antioxidant, antimutagenic, anti-inflammatory, and anticancer properties (Karimi-Khouzani et al., 2017;Limpisophon and Schleining, 2017;Silva et al., 2017;Ahmed et al., 2018). Therefore, gallic acid has been recognized as an inducer of apoptosis in cancer cell lines. It has been reported that gallic acid could induce apoptosis by ROS-dependent mitochondrial pathway in most cancer cells, such as colon cancer HCT-15 cells, small cell lung cancer H446 cells, prostate cancer DU145 cells, cervical cancer HeLa cells, melanoma A375.S2 cells (Lo et al., 2010;You et al., 2010; Subramanian et al., 2016;Wang R. et al., 2016). TC-2 ( Figure  3) is a new clerodane diterpenoid from Tinospora cordifolia (Willd.) Hook.f. & Thomson. It has been confirmed that TC-2 induced apoptosis of colon cancer cells (HCT) cells by triggering ROS production (Sharma et al., 2018). In addition, cordycepin ( Figure 3) inhibited cell growth and induced apoptosis on human brain cancer cells (SH-SY5Y and U251), related to ROS-mediated apoptosis pathway, accompanied by upregulation the expression of P53, Bax, Caspase-3/9, and downregulation the levels of Bcl-2, GPX and SOD (Chaicharoenaudomrung et al., 2018).

TTMs Targeting PI3K/Akt and JAK2/STAT3
The phosphatidylinositol-3 kinase (PI3K) signaling pathway is involved in many cancer processes. Meanwhile, the serine/ threonine specific protein kinase Akt, the main downstream effector of PI3K, is frequently activated (Chen, 2016). In addition, Akt is a key regulator of the survival, proliferation, differentiation, apoptosis, and metabolism of cancer cells. Therefore, in recent years, PIK/Akt has received considerable attention in cancer research. Signal transducer and activator of the activator of transcription 3 (STAT3) can regulate the cancer cell proliferation, apoptosis and survival by activating Janus kinase 2 (JAK-2) (Lv et al., 2016;Hoshyar and Mollaei, 2017). Rhodiola species are genera of perennial plants of the family Crassulaceae, which grow in high-altitude and cold areas in China, such as Tibet, Sichuan, Yunnan, and Qinghai (Xia et al., 2005). Among these species, Rhodiola crenulata and R. kirilowii are the most commonly used species of Hong-jing-tian as folk medicine in China. Modern studies have shown that Rhodiola species possesses a wide range of pharmacological activities, such as anti-altitude sickness, immunomodulatory, antiinflammatory, antifatigue, and anticancer activities (Kumar et al., 2010;Tao et al., 2019). It is noteworthy that R. kirilowii was reported to show potential anticancer activity. It has been found that ethanol extract of R. kirilowii in the concentration range of 10-40 mg/ml inhibited human breast cancer cells (MDA-MB-231 and HUVEC) migration and invasion, and significantly decreased phosphorylation of Akt and PKC on PI3K/Akt signaling pathway . Salidroside (Figure 3), a p-hydroxyphenethyl-b-D-glucoside, was isolated from Rhodiola species, has been reported to exhibit extensive anticancer effects. It has been verified that salidroside induced apoptosis and autophagy in human colorectal cancer cells (HT-29) through inhibition of PI3K/Akt/mTOR pathway at 0.5, 1 and 2 mM (Fan et al., 2016). In addition, salidroside also induced apoptosis in renal cell carcinoma (A498 and 786-0), and reduced the levels of p-STAT3 and p-JAK2 at a concentration of 60 mM (Lv et al., 2016). Pterocephali herba is the whole herb of the perennial plant Pterocephalus hookeri (C.B. Clarke) Höeck, a member of the Dipsacaceae family. P. hookeri has clearing heat and detoxifying functions in TTM. It is mainly used to treat rheumatoid arthritis and influenza. Recent research found that n-butanol extracts of P. hookeri with 50-200 mg/ml inhibited proliferation and induced apoptosis on Hep3B cancer cells, blocked PI3K/Akt pathway, and regulated the levels of Bcl-2 family proteins . Waltonitone (Figure 3), a pentacyclic triterpenoid of ursane type compound, was isolated from Gentiana waltonii, inhibited the cell growth, and induced apoptosis on hepatocellular carcinoma a BEL-7420 cells by modulating Akt and ERK 1/2 pathway (Zhang et al., 2009). In addition, 2-acetyl-benzylamine ( Figure 3) isolated from Justicia adhatoda L. (syn. Adhatoda vasica Nees) (0.42, 0.84, and 1.68 mM) could induce apoptosis, inhibit the expression of JAK2/ STAT3, and regulate Bcl-2/Bax ratios in MOLM-14 and NB-4 cells (Balachandran, et al., 2017).

TTMs That Downregulate the NF-kB Pathway
The nuclear factor-kappa B (NF-kB) pathway is one of the most important cellular signal transduction pathways involved in immunity, inflammation, proliferation, and apoptosis. Most of studies showed that NF-kB played a key role in cancer progression. Activation of NF-kB leads to either upregulation of antiapoptotic genes (FLIP,cIAP,survivin, or downregulation of apoptotic genes . Therefore, the combination of chemotherapy drugs with NF-kB inhibitors is considered to be an effective therapeutic strategy for the treatment of cancer. Berberis aristata, known as Ji-er-wa (Tibetan: སྐ ྱ ེ ར་པའི ་བར་ཤུ ན།) in TTM, has been widely used to treat inflammation and diabetes (Belwal et al., 2020) due to its anti-inflammatory and immunepotentiating properties. Serasanambati et al. (2015) found that different concentrations (125, 250, and 500 mg/ml) of the methanolic extracts of B. aristata could significantly inhibit cell migration and induce apoptosis in human breast cancer cells (MCF-7). Berberine and palmatine are isoquinoline alkaloids (Figure 3), which can be extracted from some medicinal plants, such as Berberis aristata, B. kansuensis, B. diaphana, B. vernae, and Coptis chinensis (Serasanambati et al., 2015;Li et al., 2018b;Neag et al., 2018;Sheng et al., 2018). Berberine exhibits multiple biologic effects with low toxicity, and the antitumor activities in various human cancer cells have been reported (Yip and Ho, 2013;Zhao et al., 2017;Yao et al., 2018). Berberine (80-160 µmol/l) induced apoptosis by suppressing NF-kB nuclear translocation via Set9-mediated lysine methylation, decreasing the levels of miR21 and Bcl-2 (Hu et al., 2012). Meanwhile, berberine (10, 50, and 100 mM) could inhibit the growth of HepG2 cells by promoting apoptosis through the NF-kB p65 pathway . It is worth noting that palmatineinduced apoptosis was associated with decreased activation of NF-kB and downstream target gene FLIP (Hambright et al., 2015).
Gentianopsis paludosa (Hook.f.) Ma is an annual Gentianaceae plant. As a traditional Tibetan medicinal material, it has been widely used as an herb in China because of its clearing heat and detoxifying functions. Lu et al. (2016) found that ethanol extract of G. paludosa could induce apoptosis of colon cancer cells (SW480), and the mechanism might be partly related to the NF-kB signaling pathway. In addition, some compounds of TTMs can also downregulate the NF-kB pathway. Cordycepin (75, 110, and 145 mmol/L) could inhibit the proliferation and induct the apoptosis of A549 cells dose-dependently, increase the expression of Bax and cleaved caspase-3, decrease the expression of Bcl-2, and the mechanism of action was achieved by inhibiting the NF-kB pathway . Choi et al. (2009) showed that gallic acid (5, 10, 25, and 50 mM) inhibited inflammatory responses caused in A549 lung cancer cells by other stimuli, including lipopolysaccharide, IFN-g, and interleukin-1b, and further downregulated the expression of NF-kB-regulated antiapoptotic genes.

TTMs That Activate AMPK Pathway
The AMP-activated protein kinase (AMPK), which is a conserved heterotrimeric protein kinase, is an important "energy sensor" regulating intracellular metabolism and energy balance and is very sensitive to changes in AMP/ATP ratio . AMPK is rapidly activated when cellular energy metabolism is abnormal, such as starvation, hypoxia, and ischemia (Ortiz et al., 2014). A series of studies have found that AMPK has strong proapoptotic potential under activated conditions. In summary, AMPK can be an important target for the treatment of cancer.
In addition to the mechanisms described above, berberine can also be used to treat cancer by activating AMPK. It was found that after berberine (12.5 and 50 mM) pretreatment of hepatocellular carcinoma cells (HepG2), the levels of p-AMPK and p-Akt were significantly increased. In addition, activation of AMPK was associated with caspase-dependent mitochondrial pathway apoptosis, coupled with mitochondrial cytochrome c release and activation of Caspase-3/9, with a dose-dependent increase in the Bax/Bcl-2 ratio. Therefore, berberine could selectively inhibit HepG2 cells' growth by inducing AMPKmediated caspase-dependent mitochondrial pathway apoptosis (Yang and Huang, 2013). Moreover, berberine (10 and 80 mM) enhanced Doxorubicin sensitivity of drug-resistant in MCF-7/ MDR breast cancer cells via AMPK/HIF-1a/P-gp pathway and directly induced apoptosis through the AMPK/p53 pathway .
Salidroside has a wide range of pharmacological activities, especially antiplateau hypoxia and immune-enhancing effects. It has been reported that salidroside can reduce superoxide dismutase (SOD) level in the mitochondria and improve endurance exercise performance. Therefore, it can be considered that salidroside reduces the production of SOD due to its effect on oxygen consumption, resulting in the change of ATP and finally the activation of AMPK. This was discovered in bladder cancer cells (UMUC3) by Liu et al. (2011). It is worth noting that salidroside could induce the autophagy-related apoptosis on human acute monocytic leukemia cells (THP-1 and U937) through AMPK activation via downregulating p62, p-PI3K, p-AKT, and p-mTOR expressions and upregulating Beclin1, LC3II and AMPK expressions (Ge et al., 2019).

CONCLUSION REMARKS
Traditional medicines are the gifts of nature to humans. Many drugs that are commonly used in modern medicine, such as artemisinin, paclitaxel, camptothecin, and ephedrine, are derived directly or indirectly from these natural medicines. TTM is an ancient health system and part of the world's traditional medical system. This system uses various treatments and personalized approaches to prevent and treat a wide range of diseases, especially chronic diseases, such as cancer.
In this review, we attempt to summarize the traditional Tibetan medical theory on the knowledge and treatment of cancer. The results showed that, in TTM, the direct cause of cancer is the shrinking and aggregating of "bad blood" owing to the reverse effect of loong (Figure 1). In addition, we review the natural Tibetan medicines traditionally used in the Tibetan system of medicine for cancer treatment. More importantly, some TTMs and their effects on apoptotic pathways are summarized in Table 4. Most TTMs exert anticancer effects through multiple components and multiple pathways. As previously mentioned, apoptosis is one of the main mechanisms by which TTM induces cancer cell death. Therefore, the molecular mechanisms of Tibetan medicine targeting apoptosis pathways are worthy of further study. However, in addition to apoptosis pathway targets, other cell death pathways may be triggered by TTMs. For example, some TTMs show anticancer activity by enhancing immunity. In order to fully evaluate the anticancer potential of these TTMs and their active ingredients, multidisciplinary approaches should be integrated to conduct pharmacological studies and reveal their mechanisms of action.
In addition, current research on TTMs is insufficient and limited. First, according to statistics (Jia and Zhang, 2016), 3,105 natural medicines have been used in the Tibetan medicine theory system. However, only 40 species have been demonstrated to possess cancer-related biological activity, and most species still lack sufficient experimental evidence. For example, brag-zhun is a natural exudate from rock stratum, which sometimes contains animal feces. Brag-zhun and its preparations are commonly used in Tibetan medicines for cancer therapy. However, to date, reports on the biological activity of the medicine associated with cancer are unavailable. Similarly, Swertia chirayita (Roxb. ex Fleming) H. Karst. and Halenia elliptica D.Don also lack cancer-related research. Given the high frequency of natural medicines being used in the treatment of cancer, supplementing these gaps in research is necessary. Secondly, although some compounds that are isolated from TTMs exhibit cancer-related biological activities, their cellular and molecular mechanisms, and possible synergies among these compounds have not been clearly elucidated. Third, TTM mainly uses prescriptions to treat cancer in clinic, but relevant research to support their application is limited. Only studies involving Yukyung Karne have been reported. Addressing these limitations in future research is necessary. Moreover, although some Tibetan herbal medicines can induce the death of cancer cells through the apoptotic pathways in vitro, these herbs have a weak anticancer effect on animal models. Therefore, in vivo experiments are necessary to verify the anticancer effects and molecular mechanisms of these TTMs.
In conclusion, this review provides the first compilation of data on TTM for cancer treatment. We found that some TTMs (e.g., O. sinensis, P. emblica, and Rhodiola kirilowii) and their active ingredients (e.g., cordycepin, salidroside, and gallic acid) have good anticancer activity. The molecular mechanisms are mainly through targeting some apoptotic pathways in cancer, for example, Bcl-2/Bax, caspases, PI3K/Akt, JAK2/STAT3, MAPK, and AMPK. These herbs and natural compounds would be potential drug candidates for cancer treatment and deserve further research and development.

AUTHOR CONTRIBUTIONS
CT and C-CZ: collected and organized the data and wrote the paper. HY and X-YW: collected the data. Z-JG: wrote the Tibetan names of natural medicines. YZ: amended the paper. YL and GF: conceived and designed the study and amended the paper.