Analysis of gene expression by reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was performed using TaqMan? Gene Expression assays (Nanog assay ID, Hs04399610_g1; SOX-2 assay ID, Hs00367969_m1; OCT4 assay ID, Hs00999632_g1; BMI1 assay ID, Hs00995536_m1; MMP9 assay ID, Hs00234579_m1; NFB1 assay ID, Hs00765730_m1; -catenin assay ID, Hs00355049_m1; CXCR4 assay ID, Hs00607978_s1; -actin assay ID, Hs01060665_g1; Thermo Fisher Scientific, Inc.) in a StepOne Real-Time PCR machine (Thermo Fisher Scientific, Inc.). more clonogenic, tumorigenic, and invasive than the corresponding depleted cells. Duhagon (13) demonstrated that TICs can be enriched using a sphere formation assay resulting in the culture of prostatospheres (PSs). Furthermore, Duhagon (13) provided a genomic profile of PSs that coordinated with the genomic profile of the MIS prostate CD44+CD24? TIC populace demonstrating that PSs are representative of the TIC populace. Klarmann (14) demonstrated that this invasive cells in the prostate G6PD activator AG1 LNCaP cell collection are more tumorigenic in NOD/SCID mice compared with noninvasive cells and have a genomic profile much like CD44+CD24? cells as well. Hence, the CD44+/CD24? cells, the PSs and the invasive cells in prostate malignancy cell lines are all representative of prostate TICs. These populations of cells express high levels of stem cell-associated genes, including OCT3/4, BMI, -catenin, and smoothened (SMO) which is usually characteristic of TICs. Additionally, TICs appear to be more resistant to standard chemotherapies and radiation, thereby, contributing to the development of metastatic and resistant disease (9,15). Given these considerations, the present study sought to investigate if prostate TICs can be targeted by Traditional Chinese Medicines (TCM) to result in the prevention of tumor initiation, progression and relapse. Herbal therapies and products generally used in TCM are bringing in increasing attention in the field of malignancy. The principles underlying TCM were established over thousands of years based on clinical experience and practice. In China, the majority of cancer patients use some form of Chinese medicine, including prescription medications and nonprescription medications (16). On a global level, it has been reported that more than half of all malignancy patients now use some form of complementary/option medicine, yet the majority of these patients do not disclose this use to their physicians (17). There are numerous clinical reports indicating that patients benefit from TCM treatment including Lin (18), which observed 173 cases of non-small cell lung malignancy (NSCLC) patients, post-surgery, with two years of treatment with standard chemoprevention alone or combined with TCM natural herbs: The result of this study indicated that this relapse and distant metastasis rate of patients in the TCM group was 45.09% and the control group was 50.6%. Yang (19) evaluated the effectiveness of comprehensive TCM treatment in reducing the relapse and metastasis of stage II and III colorectal malignancy based on standard Western medicine (WM) therapy: In this study, 222 patients were recruited and assigned to two groups based on whether or not they were additionally treated with TCM comprehensive therapy. The relapse/metastasis rate in the combined group at 1-, 2-, 3-, 4-, and 5-years was 0 (0/98), 2.04% (2/98), 11.69% (9/77), 14.06% (9/64), and 21.28% (10/47), respectively (18). In the group given WM, the relapse/metastasis rates were 4.80% (5/104), 16.35% (17/104), 21.65% (21/97), 25.93% (21/81), and 38.18% (21/55), respectively, for 1-, 2-, 3-, 4- and 5-years (19). The median relapse/metastasis time was 26.5 months in the combined group and 16.0 months in the WM group. These two studies provide a strong foundation of evidence that TCM can prohibit the relapse and metastasis of malignancy. Additionally, it has been previously shown that TCM therapy can also prevent tumorigenesis (20). Liang (21) demonstrated that this TCM Liuwei Dihuang Wan, can prohibit progression of the precancerous disease of esophageal malignancy. In this specific study, 214 patients with hyperplasia of esophageal epithelial cells were treated with Liu wei Di Huang Wan and after 2 years, the cancerous G6PD activator AG1 changes in the Liu wei Di Huang Wan treatment group was 1.4%, but in G6PD activator AG1 the placebo group was 6.3% (22). Regrettably, the active ingredients in the majority of TCM natural herbs and their mechanism(s) have not been identified. However, it is obvious that TCM is usually capable of preventing tumorigenesis and both the relapse and metastasis of malignancy (23). Previous studies have indicated that certain naturally occurring phytochemicals are cytotoxic to TICs, such as parthenolide (PTL) derived from suayule, can specifically target TICs in main human acute myelogenous leukemia (AML) (24). Additional studies exhibited that PTL has toxicity on both the side populace and mammospheres isolated from breast cancer which are representative of TICs, and lastly, Kawasaki (25) exhibited that PTL is usually cytotoxic to prostate TICs..