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Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (6): 483-494.doi: 10.1016/S2095-4964(14)60057-1

• Research Article • Previous Articles     Next Articles

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

Zhang Yuan-huia,b,c,d, Wang Yuana,b,c,d, Hussein Yusufali Alic,d, Ashby Frederickc,d, Zhang Danielc,d, Yin Zi-feia,c,d, V. Aslanidi Georgec,d,e, Srivastava Arunc,d,e,f,g, Ling Chang-quana,b, Ling Chen3,4,6   

  1. a Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai 200433, China
    b Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    c Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    d Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    e Genetics Institute, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    f Shands Cancer Center, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    g Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32611, USA
  • Received:2014-09-04 Accepted:2014-09-28 Online:2014-11-10 Published:2014-11-15

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Objective

Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted.

Methods

We synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors. 

Results

The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin (TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model. 

Conclusion

Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Key words: Medicine, Chinese traditional, Cytotoxic genes, Trichosanthin, Recombinant adeno-associated virus vector, Cancer gene therapy

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Figure 1

Schematic outline of used plasmids AAV: adeno-associated virus; AFP: alpha-fetoprotein promoter; bGH(A)n: bovine growth hormone polyA; CBAp: chicken β-actin/cytomegalovirus enhancer hybrid promoter; CMVp: cytomegalovirus promoter; EGFP: enhanced green fluorescence protein; Gluc: Gaussia luciferase; HP: hairpin structure; GOI: gene of interest; hGH(A)n: human growth hormone polyA; HP-: hairpin structure without terminal resolution site; hrGFP: humanized recombinant green fluorescence protein; IRES: internal ribosome entry site; TCS: trichosanthin."

Figure 2

Protein translations from the potential cytotoxic traditional Chinese medicine genes A: Gel electrophoresis showing Sma I-digested and undigested plasmids; B: Fluorescence microscopy results at 48 h post-transfection with indicated plasmids in HeLa cells; C: Western blot assay showing protein expression in HeLa cells."

Figure 3

Expression of cytotoxic traditional Chinese medicine genes results in a reduction in cancer cell proliferation A: Various cancer cell types were transfected with indicated plasmids. The percentage of viable cells, compared to untransfected cells, was determined by CCK-8 kit at 48 h post-transfection. Data are expressed as mean ± standard deviation; **P<0.01, vs IRES-hrGFP. B: Various hHCC cell lines were transfected with TCS-containing plasmids. Viable cell numbers were determined by CCK-8 kit over time. The red numbers indicate the transfection efficiency in various cell lines. The experiment was repeated three times using triplicate samples in each experiment. CCK-8: Cell Counting Kit-8; hHCC: human hepatocellular carcinoma; TCS: trichosanthin."

Figure 4

Effects of intracellular expressed TCS on protein translation A: Fluorescence microscopic images at 48 h post-transfection with indicated plasmids in HeLa cells. B: Expression of TCS gene dramatically decreases Gluc expression (upper panel) at 48 h post-transfection, but not the presence of Gluc mRNA (middle panel) and DNA (lower panel). The experiment was repeated three times using triplicate samples in each experiment. TCS: trichosanthin; Gluc: Guassia luciferase."

Figure 5

Expression of TCS gene induces apoptosis A: Expression of TCS genes in HeLa cells causes an increased percentage of apoptotic cells determined by annexin V/PI double staining followed by FACS analysis. Cells treated with 1 mmol/L H2O2 or etoposide for overnight were used as positive controls. B: Western blot analysis reveals that expression of TCS gene results in changes in mitochondrial pathways in HeLa cells. C: Expression of TCS gene in different human hepatocellular carcinoma cell lines causes an increase in level of cleaved PARP. 1: Mock-treated cells; 2: Cells transfected with pAAV-CMVp-IRES-hrGFP; 3: Cells transfected with pAAV-CMVp-TCS-FLAG-IRES-hrGFP; 4: Cells treated with 1 mmol/L H2O2 for 18 h. The experiment was repeated three times using triplicate samples in each experiment. TCS: trichosanthin; PARP: poly-ADP-ribose polymerase."

Figure 6

Expression of TCS gene reduces growth of hHCC xenograft in NSG mice Huh7-Fluc cells were mock-transfected or transfected with indicated plasmid, followed by subcutaneous inoculation into NSG mice. Tumor growth was then monitored over time using in vivo bioluminescent imaging. Data are expressed as mean ± standard deviation, n=5. TCS: trichosanthin; hHCC: human hepatocellular carcinoma; Fluc: firefly luciferase."

Figure 7

Intratumor delivery of TCS gene through rAAV3 vectors significantly inhibits growth of established hHCC tumor Huh7-Fluc cells were subcutaneously injected into NSG mice for 3 weeks, followed by intratumor injection of indicated rAAV3 vectors. Tumor growth was then monitored over time using in vivo bioluminescent imaging. Data are expressed as mean ± standard deviation, n=5. TCS: trichosanthin; rAAV3: recombinant adeno-associated virus serotype 3; hHCC: human hepatocellular carcinoma."

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