1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induce autophagic cell death in estrogen receptor negative breast cancer.
BACKGROUND
A novel series of methylene-substituted DIMs (C-DIMs), namely 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC6H5) groups inhibit proliferation of invasive estrogen receptor-negative MDA-MB-231 and MDA-MB-453 human breast cancer cell lines with IC50 values between 1-5 uM. The main purpose of this study was to investigate the pathways of C-DIM-induced cell death.


METHODS
The effects of the C-DIMs on apoptotic, necrotic and autophagic cell death were determined using caspase inhibitors, measurement of lactate dehydrogenase release, and several markers of autophagy including Beclin and light chain associated protein 3 expression (LC3).


RESULTS
The C-DIM compounds did not induce apoptosis and only DIM-C-pPhCF3 exhibited necrotic effects. However, treatment of MDA-MB-231 and MDA-MB-453 cells with C-DIMs resulted in accumulation of LC3-II compared to LC3-I protein, a characteristic marker of autophagy, and transient transfection of green fluorescent protein-LC3 also revealed that treatment with C-DIMs induced a redistribution of LC3 to autophagosomes after C-DIM treatment. In addition, the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker, accumulated in vacuoles after C-DIM treatment, and western blot analysis of lysates from cells treated with C-DIMs showed that the Beclin 1/Bcl-2 protein ratio increased.


CONCLUSIONS
The results suggest that C-DIM compounds may represent a new mechanism-based agent for treating drug-resistant ER-negative breast tumors through induction of autophagy.
Department of Veterinary Physiology & Pharmacology, Texas A&M University, College Station, TX 77843-4466, USA.
Kathy
K
Vanderlaag
Yunpeng
Y
Su
Arthur E
AE
Frankel
Robert C
RC
Burghardt
Rola
R
Barhoumi
Gayathri
G
Chadalapaka
Indira
I
Jutooru
Stephen
S
Safe
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D014408:Biomarkers, Tumor / Q000032:analysis*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry / Q000188:drug therapy* / Q000473:pathology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009336:Necrosis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011960:Receptors, Estrogen / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
C505817:1,1-bis(3'-indolyl)-1-(4-biphenyl)methane; C505816:1,1-bis(3'-indolyl)-1-(4-t-butylphenyl)methane; D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D014408:Biomarkers, Tumor; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D007211:Indoles; D018942:Macrolides; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C040929:bafilomycin A1; D007770:L-Lactate Dehydrogenase; D020169:Caspases