J Korean Med Sci.  2007 Sep;22(Suppl):S52-S60. 10.3346/jkms.2007.22.S.S52.

Combinational Treatment with Retinoic Acid Derivatives in Non-small Cell Lung Carcinoma In Vitro

• Choi EJ
• Whang YM
• Kim SJ
• Kim HJ
• Kim YH

• KMID: 1785789
• DOI: http://doi.org/10.3346/jkms.2007.22.S.S52

Abstract

The growth inhibitory effects of four retinoic acid (RA) derivatives, 9-cis RA, 13-cis RA, N-(4-hydroxyphenyl) retinamide (4-HPR), and all-trans retinoic acid (ATRA) were compared. In addition, the effects of various combinations of these four agents were examined on non-small cell lung carcinoma (NSCLC) cell-lines, and on the expressions of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) on these cells. At the clinically achievable concentration of 1 micrometer, only 4-HPR inhibited the growths of H1299 and H460 cells-lines. However, retinoic acid receptor beta(RAR beta) expression was up-regulated on H460 and H1299 cells treated with 1 micrometer of ATRA, 13-cis RA, or 9-cis RA. All NSCLC cell lines showed growth inhibition when exposed sequentially to 1 micrometer ATRA and 0.1 micrometer 4-HPR. In particular, sequential treatment with 1 micrometer ATRA or 13-cis RA and 4-HPR markedly inhibited H1703 cell growth; these cells exhibited no basal RAR beta expression and were refractory to 4-HPR. However, in NSCLC cell lines that expressed RAR beta, the expressional levels of RAR beta were up-regulated by ATRA alone and by sequential treatment with ATRA and 4-HPR. 4-HPR was found to be the most active of the four agents in terms of NSCLC growth-inhibition. Moreover, sequential treatments with ATRA or 13-cis RA followed by 4-HPR were found to have synergistic growth-inhibitory effects and to regulate RAR expression.

Keyword

Non-Small-Cell Lung Carcinoma; Retinoids; Chemoprevention; Fenretinide; Receptors; Retinoic Acid

MeSH Terms

Base Sequence
Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/metabolism
Cell Line, Tumor
DNA Primers/genetics
Drug Therapy, Combination
Fenretinide/administration & dosage
Gene Expression/drug effects
Humans
Isotretinoin/administration & dosage
Lung Neoplasms/*drug therapy/genetics/metabolism
Receptors, Retinoic Acid/genetics
Retinoid X Receptors/genetics
Tretinoin/administration & dosage/*analogs & derivatives

Figure

• 

  Fig. 1 RARβ mRNA expression by RT-PCR. Cells were pretreated with 4-HPR (A), 9-cis RA (B), 13-cis RA (C), or ATRA (D) at 1 µM for 48 hr. Total RNA was extracted after treating cells with each of the indicated agents for 48 hr, and the cDNA generated by MMLV reverse transcriptase was subjected to PCR using specific primers for RARβ messenger RNA (329 bp). The procedures used for total RNA isolation are described in 'Materials and Methods'. β-actin was used as an internal control. Amplified DNA was separated on 2% agarose gel and stained with ethidium bromide.

• 

  Fig. 2 Effects of combinational treatments with 4-HPR and other RAs (A), 13-cis RA (B), or 9-cis RA (C). NSCLC cell lines were treated with RAs as described in 'Materials and Methods'. Cell survivals were determined using MTT assays. Experiments were repeated 3 times to ensure reproducibility. Error bars are the means of 3 independently performed experiments and are shown with their respective standard deviations.

• 

  Fig. 3 Median effect plots of the cytotoxic effects (Fa) of sequential 13-cis RA or ATRA and 4-HPR treatment in H1703 cells. (A) H1703 cells were treated with 13-cis RA for 1 hr and then incubated for 48 hr (◆) or 72 hr (■) with 4-HPR. (B) H1703 cells were treated with ATRA for 1 hr and then incubated for 48 hr (◆) or 72 hr (■) with 4-HPR. Combination index (CI) values of <1 represent synergy between ATRA and 13-cis RA or between ATRA and 4-HPR, CI values >1 indicate antagonism and values of 1 indicate an additive effect.

• 

  Fig. 4 Northern analysis of the expressions of RARs after treating NSCLC cell lines with 4-HPR and other retinoids (ATRA, 13-cisRA, or 9-cisRA) singly or in combination. Cells were treated as described above. For Northern analysis, 10 µg of total RNA was subjected to electrophoresis in agarose gel and blotted onto a nylon membrane. H460 was a RAR positive control. GAPDH was used as an internal loading control. Blotting procedures are described in 'Materials and Methods'. Densities (%) of the RAR expressions are expressed as density ratios versus control (CT) levels.

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