An Efficient Suppression of EGFR and B-Raf mRNA Overexpression in the Lung of Benzo[a]pyrene-induced mice by Cationic Lipo-ATRA Nanoformulation.

BACKGROUND: The molecular drug all-trans retinoic acid (ATRA) acts on cancer cells via different molecular pathways, but its poor bioavailability in cancer cells limits its potency. This study was, therefore, carried out to analyse the oncogene expressions in the lung tissue of benzo[a]pyrene (B[a]P)-induced mice and compare between free ATRA and cationic liposome nanoformulation (lipo- ATRA) treatments. OBJECTIVE: This study was designed to analyse the changes in the expression levels of epidermal growth factor receptor (EGFR) and B-Raf in the lung tissues of B[a]P-induced mice during the cancer development stage itself and to find the suppressive effect of free ATRA and lipo-ATRA. METHODS: Lung cancer was induced in mice by oral ingestion of 50mg/kg body weight B[a]P weekly twice for four consecutive weeks. Then, the mice were treated with free and lipo-ATRA (0.60mg/kg) for 30 days via i.v injection. The EGFR and B-Raf gene expressions were analyzed in lung cells by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR). RESULTS: The RT-PCR gene band density and the relative quantity (RQ) values from qPCR revealed both EGFR and B-Raf genes to be significantly overexpressed in B[a]P control mice while having very low or no expression in normal mice. This indicates that they function as oncogenes in B[a]P-induced lung carcinogenesis. The lipo-ATRA treatment has shown a highly significant increase in RQ values for both EGFR and BRaf when compared to the free ATRA treatment. CONCLUSION: The study results have revealed the cationic lipo-ATRA treatment to have enhanced the bioavailability of ATRA in lung tissue due to its significant suppression action on EGFR-mediated oncogenes' expressions. Furthermore, the EGFR and BRaf could be the molecular targets of ATRA action in lung carcinogenesis.

Cytotoxic Effects of Nanoliposomal Cisplatin and Diallyl Disulfide on Breast Cancer and Lung Cancer Cell Lines.

Dual drug delivery has become the choice of interest nowadays due to its increased therapeutic efficacy in targeting the tumor site precisely. As quoted in recent literature, it has been known to treat several cancers with an acute course of action. Even so, its use is restricted due to the drug's low pharmacological activity, which leads to poor bioavailability and increases first-pass metabolism. To overcome these issues, a drug delivery system using nanomaterials which would not only encapsulate the drugs of interest but also carry them to the target site of action is needed. Given all these attributes, we have formulated dual drug-loaded nanoliposomes with cisplatin (cis-diamminedichloroplatinum(II) (CDDP)), an effective anti-cancer drug, and diallyl disulfide (DADS), an organosulfur compound derived from garlic. The CDDP and DADS-loaded nanoliposomes (Lipo-CDDP/DADS) exhibited better physical characteristics such as size, zeta potential, polydispersity index, spherical shape, optimal stability, and satisfactory encapsulation percentage. The in vitro anti-cancer activity against MDA-MB-231 and A549 cell lines revealed that Lipo-CDDP/DADS showed significant efficacy against the cancer cell lines, depicted through cell nucleus staining. We conclude that Lipo-CDDP/DADS hold exceptional pharmacological properties with better anti-cancer activity and would serve as a promising formulation to treat various cancers.

Development of optimized novel liposome loaded with 6-gingerol and assessment of its therapeutic activity against NSCLC In vitro and In vivo experimental models.

6-Gingerol (Gn) is an active compound derived from ginger which possesses various biological activities. The therapeutic applications of Gn are limited due to its hydrophobic nature. To ease its administration, one of the nano-emulsion methods, liposome was selected to encapsulate Gn. Response Surface Methodology (RSM) was used to optimize liposome ratio. 97.2% entrapment efficiency was achieved at the ratio of 1:20:2 (Drug: Lipid: Cholesterol). The optimized liposome attained size below 200 d nm, spherical shape, negative surface charge and showed sustain release upon physical characterization methods such as FESEM, DLS, Zeta potential, Drug release. The signature FTIR peaks of both free Gn and free liposome (FL) were also observed in Lipo-Gn peak. Lipo-Gn showed significant cytotoxic effect on A549 cells (IC50 160.5 ± 0.74 µM/ml) as well as inhibits the cell migration. DAPI staining showed higher apoptotic nuclear morphological change in the cells treated with Lipo-Gn, and also Lipo-Gn increased the apoptotic percentage in A549 as 39.89 and 70.32 for 12 and 24 h respectively which were significantly more than free Gn. Moreover, the formulation of Lipo-Gn showed significant cell cycle arrest at the G2/M phase compared with free Gn (28.9% and 34.9% in Free Gn vs. 42.7% and 50.1% in Lipo -Gn for 12 and 24 h respectively). Lipo-Gn have been assessed in NSCLC induced BALB/c mice and showed significantly improved pharmacological properties compared to those of free Gn. Thus, Lipo-Gn may be considered for its widening applications against lung cancer.

Synergistic effect of co-treatment with all-trans retinoic acid and 9-cis retinoic acid on human lung cancer cell line at molecular level.

The major challenge in treating cancers with ATRA is the limited availability inside the cell and resistance developed in prolonged treatment. We made an attempt for co-treatment of human NSCLC cell lines (A549) with ATRA and its isomeric precursor (9cisRA). In this study, the growth inhibitory effect of ATRA, 9cisRA and combination of both were tested in A549 cells by MTT and Trypan blue assays. As the effects of retinoid are mediated through their receptors, their gene expression levels were analyzed by RT-PCR. The target gene receptor, RAR-ß protein expression, was analyzed by immunocytochemistry. The cancer cell (A549) growth inhibitory effect was significantly (p ≤ 0.001) enhanced in combination treatment when compared with the result of individual treatments. The mRNA expression levels of both RAR-ß and RXR-ß were found to be increased in co-treatment (band density of 0.75 and 0.806, respectively) when compared with 9cisRA treatment (0.25 and 0.112) and ATRA treatment (0.01 and 0.081). A concomitant enhancement in the target RAR-ß protein expression was observed in co-treated cells when compared with individual treatments. We thus conclude that the co-treatment had increased the availability of ATRA, by isomerization of the 9cisRA which then resulted in an increased expression of both RAR-ß and RXR-ß receptors and the target protein RAR-ß which in turn inhibited lung cancer cell growth. Our study results have explored the mechanism of synergistic effect of co-treatment with ATRA and 9cisRA and further preclinical studies are necessary to validate the application of co-treatment of retinoid in clinical use.

Enhanced expression of tumour suppressor RAR-ß by DSPC nano-formulated lipo-ATRA in the lung of B16F10 cell-implanted C57BL6 mice and in A549 cells.

AIM: All Trans Retinoic acid (ATRA) is an efficient drug for leukemia, but is not efficient therapy for solid cancers. Hence we have used 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol lipo-ATRA to investigate its molecular therapeutic effect on lung cancer. The objective was to find whether it could enhance ATRA receptor, RAR-ß expression in lung cells as it was lost in majority of cancers including lung cancer. MATERIALS AND METHODS: The study was made in an experimental C57BL/6 mice model developed by tail vein injection of B16F10 cells and in A549 human lung cancer cells. The RAR-ß protein expression was studied by Immunohistochemistry/Immunocytochemistry and the mRNA expression was studied by RT-PCR and qPCR methods. KEY FINDINGS: Both free and lipo-ATRA treatments showed an enhancement of RAR-ß protein and gene expressions, indicating its induction on RAR ß. However, lipo-ATRA treatment has shown significant induction when compared with free ATRA treatment. SIGNIFICANCE: Our results implies that the DSPC lipo-ATRA treatment might have accumulated more ATRA in to the target cells which might have resulted in the induction of its receptor RAR-ß expression in a hypothesis of ligand induced receptor expression.