Chemoprevention of skin cancer with 1,1-Bis (3'-indolyl)-1-(aromatic) methane analog through induction of the orphan nuclear receptor, NR4A2 (Nurr1).
BACKGROUND
The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3'-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.


METHODS
In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.


RESULTS
The IC50 values of DIM-D were 68.7 ± 7.3, 48.3 ± 10.1 and 11.5 ± 3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1 ± 8.3, 186.1 ± 5.2 and 56.7 ± 3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.


CONCLUSIONS
Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, Florida, United States of America.
Cedar H A
CH
Boakye
Ravi
R
Doddapaneni
Punit P
PP
Shah
Apurva R
AR
Patel
Chandraiah
C
Godugu
Stephen
S
Safe
Santosh K
SK
Katiyar
Mandip
M
Singh
D000080242:8-Hydroxy-2'-Deoxyguanosine; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018890:Chemoprevention / Q000379:methods; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects* / Q000528:radiation effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D014466:Ultraviolet Rays
C534880:1,1-bis(3'-indolyl)-1-(4-chlorophenyl)methane; D007211:Indoles; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D017382:Reactive Oxygen Species; D000080242:8-Hydroxy-2'-Deoxyguanosine; D002392:Catechin; C045651:epigallocatechin gallate; D003849:Deoxyguanosine