Chapter Four: Critical Analysis of Scientific Article (I)

Delta-9-Tetrahydrocannibinol Inhibits Epithelial Growth-Factor-Induced Lung Cancer Cell Migration in Vitro as Well as Its Growth and Metastasis in Vivo

This post will introduce you to the first of two anyleses on research articles that focus on specific types of cancer. The first article introduces the topic of THC in the treatment of lung cancer. It will be broken down to better dissect the research article. 

Main Research Question

• Effects of THC on the EGF-induced growth and metastasis on human cell lung cancer in vitro (meaning in the laboratory)
• Effects of THC on tumor growth and lung metastasis in vivo (meaning inside an organism) 

Methods

• Western Blot and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
• Examined expression of cannabinoid receptor in human NSCLC (Non-Small Cell Lung Cancer) cell lines
• Cell Line #1 - A549
• Cell Line #2 - SW-1573
• Examined expression of EGFR (Epithelial Growth Factor Receptor) in human NSCLC cell lines
• Treatment with THC (In Vitro)
• Examined effect of THC on cell migration/motility (EGF and EGFR play important roles in cell migration)
• Percent colonization of the wound areas in cancer cell lines compared to vehicle-treated EGF-stimulated wounds (scratch wound assay)
• Tryptan Blue Staining
• Examined cell viability
• Treatment with THC (In Vivo)
• Intravenous injection through lateral tail vein of SCID (severe combined immunodeficient) mice
• Injection of THC (or vehicle) peritumorally daily for 21 says
• Mice Tumor Sample Analysis
• Analyzed cell proliferation, vascularization, and phosphorylation of signaling molecules FAK, ERK1, ERK2, and AKT

Results

• Western Blot and RT-PCR
• Expressed CB1 and CB2 receptors
• Treatment with THC (In Vitro)
• THC induced cell rounding which led to failure of the cell to produce characteristic protrusions
• No effect on viability of cells
• Induce apoptosis and inhibit proliferation
• Decreased EGF-stimulated cell migration
• Inhibited EGF-induced transwall migration in a dose-dependent manner
• No effect on EGFR expression or phosphorylation
• Enhanced EGF-induced phosphorylation of FAK at tyrosine 397 and inhibition of AKT phosphorylation
• Inhibited EGF-induced invasion in dose-dependent manner 
• Tryptan Blue Staining
• Concentration of THC did not have any significant effect on viability of cell lines
• Treatment with THC (In Vivo)
• Surface lung metastases were significantly reduced
• Significantly reduced tumor weight and number lesions
• Tumor growth in THC-treated animals was inhibited compared to that in vehicle-treated animals
• No significant alterations in physiological parameters like body or liver weight were observed
• Mice Tumor Sample Analysis
• Inhibit in vivo tumor cell proliferation and vascularization determined by Ki67 and CD31 immunostaining
• Phosphorylation of FAK, ERK 1, ERK 2, and AKT were reduced in THC-treated tumors 

Analysis of Results (Discussion)

• Comparison to Previous Literature
• Previous studies demonstrated tumor-promoting or antineoplastic effects of THC, but no research is on the effect of EGFR-mediated growth and motility of lung cancer
• Expression of CB2 receptors in A549 cells was questioned, but this study confirmed CB1 and CB2 expression on both A549 and SW-1573
• THC treatment attenuated EGF-induced morphological changes like cell elongation and generation of protrusions leading to rounding and reduction of mobility
• Antimigratory effect of THC was previously found
• Advancement in Scientific Knowledge
• EGFR-mediated activation of MAP kinases has reported to regulate EGF-induced cell migration and invasion – this study observed a reduction in EGF-induced ERK1/2 and JNK1/2 phosphorylation
• Other studies have reported THC-induced activation of AKT/PKB – this study found that THC reduced AKT phosphorylation induced by EGF
• THC exhibits equal affinity toward CB1 and CB2
• The high case:fatality ratio observed in lung cancer is attributed to a poor response to therapy and the aggressive biological nature of the disease
• High expression of Epidermal Growth Factor Receptor (EGFR) is common in non-small cell Lung Cancer (NSCLC) and correlates to a more aggressive disease, resistance to chemotherapy, and poor prognosis
• Additional Questions
• Molecular mechanism involved in THC-mediated inhibition of chemotaxis induced by EGF are not well characterized
• Modulation of EGFR expression with THC
• Function of FAK, ERK 1/2, and AKT in tumors

Molecular Biology Techniques Used in Experiment

• Western Blot: analytical technique that detects specific proteins in a sample that is extracted from a tissue sample. Using gel electrophoresis, the proteins in the sample are separated by molecular weight using a current that passes through the gel, which pulls smaller proteins further down the gel and leaves larger proteins closer to the top of the gel (wells)
• Polymerase Chain Reaction (PCR): method of amplifying copies of a gene on DNA. Typically it involves denaturing of the DNA strand (using heat to separate the strands) followed by the annealing (binding) of a primer that is complimentary to a region of the nucleotide strand, and lastly the the elongation of the strand via DNA polymerase (enzyme that adds nucleotides to already-existing nucleotide strand
• Tryptan Blue Staining: visualization technique that selectively dyes dead tissues or cell the color blue, usually used to assess the tissue viability

The entire article (with images!) can be found here:

http://www.readcube.com/articles/10.1038/sj.onc.1210641

References

Preet, A., Ganju, R., & Groopman, J. (2007) Delta-9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell mgiration in vitro as well as its growth and metastasis in vivo. Oncogene. 27(3): 339-46.