Recipe Of The Screening Assessment Of Chinese Medicine Treatment Of Liver Cancer Study

Hepatocellular carcinoma (HCC) is a common form of solid tumor in human and its occurrence is rapidly increasing globally. HCC is characterized by its unclear syndrome, rapid progression, malignance, metastasis, recurrence and low survival rate. According to the World Health Organization, worldwide death caused by HCC-related diseases has gone up to one million annually. Up to date, surgery remains to be the major clinical treatment. Only 40% of the patient suffering from HCC, however, may be benefited from surgery. Most patients are excluded from surgery due to their health conditions, hepatic function, location and/or size of the tumors, cirrhosis, serious jaundice or ascites. Besides, a 40% to 60% rate of recurrence has been reported to patients who accepted surgery.Neither invasive treatment, nor chemotherapeutic drugs have shown satisfactory results for HCC treatment. In addition, the side-effect and toxicity of the chemotherapy have also discouraged many patients who may give up the clinical treatment. Although TCM practices based on differentiation of patterns and subsequent treatment have shown its effects and advantages in HCC clinical treatment. It's still not scientifically proven and hence TCM remains to be non-comparable with the main-stream, modern western medical practices.ObjectivesCurrent standard TCM practices based on differentiation of patterns are insufficient in treating HCC that is so complicated clinically and physiologically. Observation of the progress of HCC, together with treatment (principles and protocols), has therefore been difficult. TCM research on HCC has focused on physiological mechanisms, lending few guidance for TCM clinical formula composition, nor criteria for effectiveness. A comparable TCM to western medicine is critically needed for HCC clinical research.Application of modern biotechnology to TCM has become a new approach for a) TCM formula composition and combination, plus scientific validation; b) review and validation of treatment principles and protocols; c) study on the occurrence of HCC; d) study on the TCM differentiation of patterns; e) criteria for clinical evaluation. This combination and scientific approach (method and technical platforms) may benefit both TCM and Western medicine practices for HCC. It's also the objectives of this study in evaluating the TCM clinical medicines, improving their effects, as well as screening for a better formula composition.Experimental TCM samplesQuite a few TCM single ingredients, formula composition are available in the literatures. Most of them were clinically evidenced, yet few were scientifically validated. Conventional TCM considers "heat toxin" as one of the causes for tumor formation. Formula that releases heat and detoxify may inhibit tumors have also been suggested by some clinical evidences] and the author's practice experiences.A search for formula composition, excluding those prohibited by Health Canada, and protected by CITES, resulted in 11 samples including seven formula compositions together with single ingredients of the formula. These are Sample A,Sample B,Sample C,Sample D,Sample F,Sample G,Sample H,Sample J, Sample K,Sample L,Sample M. Among them, the Sample C has been a formula for hepatitis at the author's clinic. Experimental MethodsThis study involves an in-vitro screening test for some anti-tumor traditional Chinese medicine (TCM) preparations. TCM preparations in the format of either single or combination formulation were selected to observe its anti-tumor activity, as well as its effect on the physiology of tumor cell lines HegG2 and HL-60. Results of these in-vitro tests (MTT reducing method, single cell microfluid chip) were compared with the author's clinical data, practices experiences along with the Chinese medicine theories. Two cell lines, human hepatic cell line HepG2 and human leukemia cell line HL-60, were selected for the cell culture required for in-vitro test studies, which applied the conventional MTT reducing method and a novel microfluid single-cell biochip as platforms. Using the MTT reducing test, one can study the survival rate of cells under the treatment of bioactive compounds. In other words, one can study the anti-tumor activity of a bioactive compound.The microfluid biochip (MFB) is able to "retain" a single cell in a chamber, while maintaining its biological and physiological activities.Its design also allows a bioactive compounds (dissolved in a microfluid) to flow through the retained, live cell, and to be metabolized by the cell. The monitoring of cellular metabolism took advantage of the correlation between the cellular calcium ion concentration and the cellular physiological activity. The detector of the MFB system applies a photometric mechanism to detect and record the variation of the calcium ion concentration, which, indirectly, indicates the straightforward effect of the bioactive compound on the single cell. By using this MFB platform, one can achieve a "real time" drug metabolism study or a cytotoxic evaluation at a cellular level. Results from these in-vitro screen tests were reviewed and compared with TCM theory on formula composition. The derived formula will then lead to clinical guidelines and protocols.Experimental ResultsAccording to the MTT test results, four TCM formula composition including sample B(加味柴胡清肝汤I), sample C(加味柴胡清肝汤Ⅱ),sample F(茵陈五苓散),sample H(清肝抗癌方)showed a concentration-correlated inhibition of HepG2 cell-line. Among them, sample H was statistically significant. Sample K(斑蝥) showed the highest effect on inhibiting HepG2 among the ingredients. However, its inhibition effect was not correlated with a strong ca++ ion activity as suggested by the single cell chip experiment. Hence, its effect on cellular metabolism was not conclusive. Sample M (金银花) affected the ca++ ion activity of both cell lines under test. Sample L (白花蛇舌草) did not trigger ca++ ion activity of HepG2 cell line, but seemed affect HL-60 cell line. Sample H also affected the ca++ ion activity of both cell lines under test. Samples B, C and D, the formula that were supplemented, all showed anti-tumor effect, whereas Sample A, Sample F and Sample G did not show significant effect.Discussion and ConclusionExperimental results suggest that sample H inhibited cell growth of both cell lines, indicating its potential as anti-tumor agents. Its formula composition (Sample J, K, L, M) when compared with TCM theory of formulation functions, was also well verified. Except sample K, none of the single ingredient had shown significant cell-growth inhibition. A formula composition of these ingredients, if effective, may only be explained by a synergistic effect. On the other hand, TCM clinical practice, based on the principle of "diagnosis via differentiation of patterns and treatment via references", may modify the formula as needed. The fact that all samples are commercially available powder products suggests their safety and efficacy, and is therefore clinically significant. Further proof may have to come from animal models, safety and toxicity test. Further study of the single cell metabolism upon synergetic effect may require little modification of the biochip designs. There has no consensus about the cause for HCC yet. It may be a combinatory result of many factors, including Hepatitis B virus, Hepatitis C virus, cancerous chemical pollutants such as aflatoxins, dichloroethane, nitrosamines, thorium dioxide, parasites (Schistosoma japonicum, liver fluke), plus stress, emotional impacts, immune system disorder, cellular antigen-related functions. Clinical observations include HbsAg positive results (80-90% to cancer patients), cirrhosis, cholangitis, gall stone suggest a three step prognosis "from hepatitis to cirrhosis and to tumors" that is in line with TCM theory about HCC - "HCC is from an accumulated moisture, heat, stasis and toxin". TCM theory therefore provides guidance for the clinical formula composition, which was proven in this study. CommentsApplying modern medical sciences, in particular, the advancing biotechnology to the screening of TCM drug formula, to clinical TCM drug compounding is a scientific validation approach. This study attempted such an approach for the evaluation of a clinically useful TCM formula composition, and proved that modern biotechnical platforms can be combined with traditional TCM drug screening research. Well-known TCM expert doctor Tie-Tao Deng(邓铁涛)commented that in the course of development of TCM, the first milestone was invention of formula compositions. Subsequently, the second milestone was various guidelines and protocols. And the third milestone will be the TCM Theory combined with modern biotechnology for coming years.