Lung cancer metastasis has no direct treatment
Lung cancer is the primary cause of cancer related deaths worldwide in men and the second highest cause for women. In total lung cancer accounts for approximately 25% of all cancer related deaths. Of all the types of lung cancer the non small cell lung cancer (NSCLC) is the most common form and accounts for 85% of reported cases.
Cancer metastasis is the primary reason for treatment failure and the death of lung cancer patients. During metastasis, cancer cells break off from where they first formed (primary cancer), travel through the blood or lymph system, and form new tumors (metastatic tumors) in other areas of the body. These metastatic tumors are of the same type of cancer as the primary tumor.
The accepted standard of care is chemotherapy, which carries a host of problems itself, but unfortunately there is currently no effective method to directly target metastasis in lung cancer.
However, recently progress was made in understanding the underlying mechanism behind lung cancer metastasis. It was reported that epithelial-to-mesenchymal transition (EMT) plays a key role in cancer movement and migration during metastasis. EMT performs a variety of functions and leads to the disconnection of cell to cell adhesion. Transforming growth factor-β (TGF-β) is a known promoter of EMT in cancer cells during tumor growth (1). Tumor necrosis factor-α (TNF-α) is also well documented as being able to enhance the EMT process initiated by TGF-β (2).
Honokiol and metastasis
Today we are going to have a look at an interesting study from September 2016 where the researchers investigated the connection between EMT, TGF-β and TNF-α and the use of honokiol as a potential way to inhibit this process to prevent metastasis (3).
The researchers knew that honokiol had been used to inhibit EMT in other kinds of cancer and they wanted to see if the same effect would be seen in lung cancer cells. The first step was using a combination of TGF-β1 and TNF-α in two human non-small cell lung cancer (NSCLC) cells lines to induce the EMT process. TNF-α and TGF-β1 had already been used to stimulate EMT in other cancer cells in previous studies (4-5) so the research team were confident it would work the same for lung cancer cells.
Honokiol reduces cancer cell proliferation
The research team stimulated EMT and examined the level of cell proliferation, cell motility (movement) and protein expression in the cells. The cancer cell lines (A549 and H460) were exposed to 25 ng/mL TNF-α and 5 ng/mL TGF-β1 and combined with honokiol in various doses as shown in the diagrams below.
The results showed that honokiol was able to reduce the growth of both cell lines in a dose-dependent manner confirming what other researchers had previously reported (6). Not only this but TNF-α and TGF-β1 in combination with honokiol increased the inhibition of A549 cell proliferation, but there was no significant difference on H460 cells between the three combined or honokiol alone. In all cases honokiol was able to reduce the rate of cancer cell proliferation, based on the results the researchers chose 30 μmol/L of honokiol combined with 25 ng/mL of TNF-α and 5 ng/mL of TGF-β1 as optimal for further tests.
Honokiol can inhibit the migration of cancer cells
Previous studies have shown that honokiol inhibits the migration of lung cancer cells by targeting β-catenin signaling activation mediated by PGE2 (7). The researchers again combined TNF-α, TGF-β1 and honokiol and examined the inhibition of motility and reduction of migration in cancer cell lines using a wound-healing assay and a co-culture system.
The researchers found that in their wound-healing assay, treatment with TNF-α and TGF-β1 resulted in a decrease in the size of the wound area due to movement of A549 and H460 cells to the scarred region compared to the control. However in cells treated with honokiol induced with TNF-α TGF-β1 cell migration was inhibited. In a similar manner during the co-culture assay, TNF-α+TGF-β1 treatment resulted in a high level of cell migration, TNF-α+TGF-β1 and honokiol significantly lower and honokiol alone was lower still.
Honokiol affects the expression of EMT-associated proteins and c-FLIP
Cellular FLICE-inhibitory protein (c-FLIP) is a master anti-apoptotic regulator and resistance factor that suppresses tumor necrosis factor-α (TNF-α) and influences the expression of N-cadherin (a protein involved in metastasis) and snail (a key inducer of EMT that also plays an important role in cell survival). As the researchers previously showed, honokiol modulates the expression level of c-FLIP whilst inhibiting the migration and motility of lung cancer cells.
The research team next explored the relationship between EMT and c-FLIP and the first step was to monitor expression of N-cadherin, snail and c-FLIP after exposure to honokiol or TNF-α and TGF-β1. The team found that TNF-α and TGF-β1 increased expression of N-cadherin, snail, and c-FLIP and that honokiol significantly decreased them.
Further experimentation confirmed that the expression of c-FLIP was directly correlated with the movement and migration of the cancer cells and that it regulates the expression of N-cadherin and snail. They also discovered that the downstream effectors of c-FLIP were NF-κB (a protein complex involved in regulating the inflammatory response) and N-cadherin and snail. The researchers also suggested based on their results, that Smad signaling may be upstream of c-FLIP.
In closing, the researchers here show a mechanism where honokiol prevents EMT mediated migration and motility of lung cancer cells via inhibition of c-FLIP. This makes c-FLIP a promising target for cancer research and that honokiol with its various anticancer properties holds potential as a drug to prevent metastasis for lung cancer.
(1)Li, L., Qi, L., Liang, Z., Song, W., Liu, Y., Wang, Y., … & Cao, W. (2015). Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells. International journal of molecular medicine, 36(1), 113-122.
(2)Saito, A., Suzuki, H. I., Horie, M., Ohshima, M., Morishita, Y., Abiko, Y., & Nagase, T. (2013). An integrated expression profiling reveals target genes of TGF-β and TNF-α possibly mediated by microRNAs in lung cancer cells. PloS one, 8(2), e56587.
(3)Lv, X. Q., Qiao, X. R., Su, L., & Chen, S. Z. (2016). Honokiol inhibits EMT-mediated motility and migration of human non-small cell lung cancer cells in vitro by targeting c-FLIP. Acta Pharmacologica Sinica, 37(12), 1574-1586.
(4)Maier, H. J., Schmidt-Straßburger, U., Huber, M. A., Wiedemann, E. M., Beug, H., & Wirth, T. (2010). NF-κB promotes epithelial–mesenchymal transition, migration and invasion of pancreatic carcinoma cells. Cancer letters, 295(2), 214-228.
(5)Takahashi, E., Nagano, O., Ishimoto, T., Yae, T., Suzuki, Y., Shinoda, T., … & Tanihara, H. (2010). Tumor necrosis factor-α regulates transforming growth factor-β-dependent epithelial-mesenchymal transition by promoting hyaluronan-CD44-moesin interaction. Journal of Biological Chemistry, 285(6), 4060-4073.
(6)Lv, X., Liu, F., Shang, Y., & Chen, S. Z. (2015). Honokiol exhibits enhanced antitumor effects with chloroquine by inducing cell death and inhibiting autophagy in human non-small cell lung cancer cells. Oncol Rep, 34, 1289-1300.
(7)Singh, T., & Katiyar, S. K. (2013). Honokiol inhibits non-small cell lung cancer cell migration by targeting PGE 2-mediated activation of β-catenin signaling. PloS one, 8(4), e60749.