Eun Ah Shin

Melatonin Suppresses the Expression of 45S Preribosomal RNA and Upstream Binding Factor and Enhances the Antitumor Activity of Puromycin in MDA-MB-231 Breast Cancer Cells

Since the dysregulation of ribosome biogenesis is closely associated with tumor progression, in the current study, the critical role of ribosome biogenesis related signaling was investigated in melatonin and/or puromycin induced apoptosis in MDA-MB-231 breast cancer cells. Despite its weak cytotoxicity, melatonin from 3 mM attenuated the expression of 45S pre-ribosomal RNA (pre-rRNA), UBF as a nucleolar transcription factor, and fibrillarin at mRNA level and consistently downregulated nucleolar proteins such as UBF and fibrillarin at protein level in MDA-MB-231 cells. Furthermore, immunofluorescence assay revealed that UBF was also degraded by melatonin in MDA-MB-231 cells. In contrast, melatonin attenuated the expression of survival genes such as Bcl-xL, Mcl-1, cyclinD1, and cyclin E, suppressed the phosphorylation of AKT, mTOR, and STAT3, and cleaved PARP and activated caspase 3 only at a high concentration of 12 mM. However, combined treatment of melatonin (3 mM) and puromycin (1 μM) synergistically inhibited viability, attenuated the expression of 45S pre-rRNA and UBF, and consistently downregulated UBF, XPO1 and IPO7, procaspase 3, and Bcl-xL in MDA-MB 231 cells. Overall, these findings suggest that melatonin can be a cancer preventive agent by combination with puromycin via the inhibition of 45S pre-rRNA and UBF in MDA-MB 231 breast cancer cells.

c-Jun N-terminal Kinase-Dependent Endoplasmic Reticulum Stress Pathway is Critically Involved in Arjunic Acid Induced Apoptosis in Non-Small Cell Lung Cancer Cells.

Though arjunic acid, a triterpene isolated from Terminalia arjuna, was known to have antioxidant, antiinflammatory, and cytotoxic effects, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the molecular antitumor mechanism of arjunic acid was examined in A549 and H460 non‐small cell lung cancer (NSCLC) cells. Arjunic acid exerted cytotoxicity by 3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl tetrazolium bromide (MTT) assay and significantly increased sub‐G1 population in A549 and H460 cells by cell cycle analysis. Consistently, arjunic acid cleaved poly (ADP‐ribose) polymerase (PARP), activated Bax, and phosphorylation of c‐Jun N‐terminal kinases (JNK), and also attenuated the expression of pro‐caspase‐3 and Bcl‐2 in A549 and H460 cells. Furthermore, arjunic acid upregulated the expression of endoplasmic reticulum (ER) stress proteins such as IRE1 α, ATF4, p‐eIF2α, and C/EBP homologous protein (CHOP) in A549 and H460 cells. Conversely, CHOP depletion attenuated the increase of sub‐G1 population by arjunic acid, and also JNK inhibitor SP600125 blocked the cytotoxicity and upregulation of IRE1 α and CHOP induced by arjunic acid in A549 and H460 cells. Overall, our findings suggest that arjunic acid induces apoptosis in NSCLC cells via JNK mediated ER stress pathway as a potent chemotherapeutic agent for NSCLC.

Apoptotic Effect of Galbanic Acid via Activation of Caspases and Inhibition of Mcl-1 in H460 Non-Small Lung Carcinoma Cells.

Galbanic acid (GBA), a major compound of Ferula assafoetida, was known to have cytotoxic, anti‐angiogenic and apoptotic effects in prostate cancer and murine Lewis lung cancer cells; the underling apoptotic mechanism of GBA still remains unclear so far. Thus, in the present study, the apoptotic mechanism of GBA was investigated mainly in H460 non‐small cell lung carcinoma (NSCLC) cells because H460 cells were most susceptible to GBA than A549, PC‐9 and HCC827 NSCLC cells. Galbanic acid showed cytotoxicity in wild EGFR type H460 and A549 cells better than other mutant type PC‐9 and HCC827 NSCLC cells. Also, GBA significantly increased the number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells and sub G1 population in H460 cells. Western blotting revealed that GBA cleaved poly (ADP‐ribose) polymerase (PARP), activated Bax and caspase 9, attenuated the expression of Bcl‐2, Bcl‐xL, and Myeloid cell leukemia 1 (Mcl‐1) in H460 cells. However, interestingly, overexpression of Mcl‐1 blocked the ability of GBA to exert cytotoxicity, activate caspase9 and Bax, cleave PARP, and increase sub G1 accumulation in H460 cells. Overall, these findings suggest that GBA induces apoptosis in H460 cells via caspase activation and Mcl‐1 inhibition in H460 cells as a potent anticancer agent for NSCLC treatment. Copyright

Auraptene Induces Apoptosis via Myeloid Cell Leukemia 1‐Mediated Activation of Caspases in PC3 and DU145 Prostate Cancer Cells

Although auraptene, a prenyloxy coumarin from Citrus species, was known to have anti‐oxidant, anti‐bacterial, antiinflammatory, and anti‐tumor activities, the underlying anti‐tumor mechanism of auraptene in prostate cancers is not fully understood to date. Thus, in the present study, we have investigated the anti‐tumor mechanism of auraptene mainly in PC3 and DU145 prostate cancer cells, because auraptene suppressed the viability of androgen‐independent PC3 and DU145 prostate cancer cells better than androgen‐sensitive LNCaP cells. Also, auraptene notably increased sub‐G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling‐positive cells as features of apoptosis in two prostate cancer cells compared with untreated control. Consistently, auraptene cleaved poly(ADP‐ribose) polymerase, activated caspase‐9 and caspase‐3, suppressed the expression of anti‐apoptotic proteins, including Bcl‐2 and myeloid cell leukemia 1 (Mcl‐1), and also activated pro‐apoptotic protein Bax in both prostate cancer cells. However, Mcl‐1 overexpression reversed the apoptotic effect of auraptene to increase sub‐G1 population and induce caspase‐9/3 in both prostate cancer cells. Taken together, the results support scientific evidences that auraptene induces apoptosis in PC3 and DU145 prostate cancer cells via Mcl‐1‐mediated activation of caspases as a potent chemopreventive agent for prostate cancer prevention and treatment. Copyright

Caspase inhibitors: a review of recently patented compounds (2013-2015)

ABSTRACT Introduction: Although many caspase inhibitors have been patented, caspase inhibitors have not entered the market due to their toxicity and poor pharmacokinetic profile. Areas covered: In this article, we review patents (2013–2015) for peptide and non-peptide caspase inhibitors and their compositions. Expert opinion: Noteworthy patents include a peptidic caspase-2 inhibitor for nasal administration and a peptidomimetic caspase-6 inhibitor that can be administered via several routes for the treatment of neurodegenerative diseases. Furthermore, caspase-1 inhibitors for contact dermatitis and inflammation, cardiovascular diseases, and liver diseases and a caspase-3 inhibitor for cerebral stroke have been patented. Of particular interest is the novel use of tyrosine kinase inhibitors (sunitinib and its derivatives) for the prevention and treatment of age-related ocular diseases via inhibition of the caspase-3, dual-leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK) pathways. However, for effective clinical application of caspase inhibitors, novel peptidic and nonpeptidic caspase inhibitors with lower toxicity and improved efficacy should be developed via structural modifications, and further animal studies and preclinical and clinical trials are needed. In addition, the poor pharmacokinetic properties of classic caspase inhibitors may be improved by using advanced drug delivery systems that employ liposomes, polymers, and nanoparticles through effective administration routes.

Obovatol Induces Apoptosis in Non-small Cell Lung Cancer Cells via C/EBP Homologous Protein Activation

Although obovatol, a phenolic compound from the bark of Magnolia obovata, was known to have antioxidant, neuroprotective, antiinflammatory, antithrombotic and antitumour effects, its underlying antitumour mechanism is poorly understood so far. Thus, in the present study, the antitumour molecular mechanism of obovatol was investigated in non‐small cell lung cancer cells (NSCLCs). Obovatol exerted cytotoxicity in A549 and H460 NSCLCs, but not in BEAS‐2B cells. Also, obovatol increased sub‐G1 accumulation and early and late apoptotic portion in A549 and H460 NSCLCs. Consistently, obovatol cleaved PARP, activated caspase 9/3 and Bax and attenuated the expression of cyclin D1 in A549 and H460 NSCLCs. Interestingly, obovatol upregulated the expression of endoplasmic reticulum stress proteins such as C/EBP homologous protein (CHOP), IRE1α, ATF4 and p‐elF2 in A549 and H460 NSCLCs. Conversely, depletion of CHOP blocked the apoptotic activity of obovatol to increase sub‐G1 accumulation in A549 and H460 NSCLCs. Overall, our findings support scientific evidences that obovatol induces apoptosis via CHOP activation in A549 and H460 NSCLCs. Copyright

Apoptotic Effect of Astragalin in Melanoma Skin Cancers via Activation of Caspases and Inhibition of Sry-related HMg-Box Gene 10

Though Astragalin (kaempferol‐3‐glucoside) contained in Paeonia lactiflora and other plants was known to have anti‐oxidant, antiinflammatory, and anti‐tumor activity, the anti‐tumor mechanism of Astragalin has never been reported in melanomas until now. Thus, in the present study, the underlying apoptotic mechanism of Astragalin isolated from Aceriphyllum rossii was elucidated in A375P and SK‐MEL‐2 melanoma cells. Astragalin exerted cytotoxicity in A375P and SK‐MEL‐2 cells in a concentration‐dependent manner. Also, Astragalin significantly increased the number of TdT‐mediated dUTP nick end labeling positive cells and sub‐G1 population as a feature of apoptosis in A375P and SK‐MEL‐2 cells compared with untreated control. Consistently, western blotting revealed that Astragalin activated caspase 9/3 and Bax, cleaved poly (ADP‐ribose) polymerase, and attenuated the expression of cyclin D1, Mcl‐1, and Sry‐related HMg‐Box gene 10 (SOX10) in A375P and SK‐MEL‐2 cells. Of note, ectopic expression of SOX10 reduced the apoptotic ability of Astragalin to inhibit proliferation, cleave poly (ADP‐ribose) polymerase, and caspase 3 in A375P and SK‐MEL‐2 melanoma cells. Overall, our findings provide evidence that Astragalin induces apoptosis in A375P and SK‐MEL‐2 melanoma cells via activation of caspase9/3 and inhibition of SOX10 signaling. Copyright

Implications of Bcl-2 and its interplay with other molecules and signaling pathways in prostate cancer progression

ABSTRACT Introduction: Among several genetic alterations involved in the progression of prostate cancer, B cell lymphoma gene number 2 (BCL-2) is an important target molecule in the progression of androgen-independent prostate cancer (AIPC) after androgen ablation or castration. Nevertheless, the molecular mechanism of BCL-2 in prostate cancer progression remains elusive and controversial. In the current review, we discuss the critical role of BCL-2 in the carcinogenesis of prostate cancer with experimental evidences on the BCL-2 molecular networks in AIPC and androgen-dependent prostate cancer (ADPC) and subsequently suggest perspective research targeting BCL-2. Areas covered: This review focused on the molecular implications of BCL-2 in association with other molecules and signaling pathways involved in the progression and carcinogenesis of prostate cancer. Expert opinion: BCL-2 plays a pivotal role in the progression of AIPC than in ADPC since androgen represses BCL-2. BCL-2 acts as a pro-survival molecule in association with androgen-related signaling in the progression of ADPC, while BCL-2 upregulation, PTEN loss, PI3K/AKT phosphorylation and receptor tyrosine kinase (RTK) activation are primarily involved in AIPC. To identify more effective prostate cancer therapy, further mechanistic studies are required with BCL-2 inhibitors in AIPC and ADPC, considering a multi-target therapy against BCL-2 and its related signaling.

Melatonin disturbs SUMOylation-mediated crosstalk between c-Myc and nestin via MT1 activation and promotes the sensitivity of paclitaxel in brain cancer stem cells

Here the underlying antitumor mechanism of melatonin and its potency as a sensitizer of paclitaxel was investigated in X02 cancer stem cells. Melatonin suppressed sphere formation and induced G2/M arrest in X02 cells expressing nestin, CD133, CXCR4, and SOX‐2 as biomarkers of stemness. Furthermore, melatonin reduced the expression of CDK2, CDK4, cyclin D1, cyclin E, and c‐Myc and upregulated cyclin B1 in X02 cells. Notably, genes of c‐Myc related mRNAs were differentially expressed in melatonin‐treated X02 cells by microarray analysis. Consistently, melatonin reduced the expression of c‐Myc at mRNA and protein levels, which was blocked by MG132. Of note, overexpression of c‐Myc increased the expression of nestin, while overexpression of nestin enhanced c‐Myc through crosstalk despite different locations, nucleus, and cytoplasm. Interestingly, melatonin attenuated small ubiquitin‐related modifier‐1 (SUMO‐1) more than SUMO‐2 or SUMO‐3 and disturbed nuclear translocation of nestin for direct binding to c‐Myc by SUMOylation of SUMO‐1 protein by immunofluorescence and immunoprecipitation. Also, melatonin reduced trimethylated histone H3K4me3 and H3K36me3 more than dimethylation in X02 cells by Western blotting and chromatin immunoprecipitation assay. Notably, melatonin upregulated MT1, not MT2, in X02 cells and melatonin receptor inhibitor luzindole blocked the ability of melatonin to decrease the expression of nestin, p‐c‐Myc(S62), and c‐Myc. Furthermore, melatonin promoted cytotoxicity, sub‐G1 accumulation, and apoptotic body formation by Paclitaxcel in X02 cells. Taken together, these findings suggest that melatonin inhibits stemness via suppression of c‐Myc, nestin, and histone methylation via MT1 activation and promotes anticancer effect of Paclitaxcel in brain cancer stem cells.

Farnesiferol c induces apoptosis via regulation of L11 and c-Myc with combinational potential with anticancer drugs in non-small-cell lung cancers.

Though Farnesiferol c (FC) has been reported to have anti-angiogenic and antitumor activity, the underlying antitumor mechanism of FC still remains unclear. Thus, in the present study, we investigated the apoptotic mechanism of FC in human H1299 and H596 non-small lung cancer cells (NSCLCs). FC significantly showed cytotoxicity, increased sub-G1 accumulation, and attenuated the expression of Bcl-2, Bcl-xL, Survivin and procaspase 3 in H1299 and H596 cells. Furthermore, FC effectively suppressed the mRNA expression of G1 arrest related genes such as Cyclin D1, E2F1 transcription factor and CDC25A by RT-PCR. Interestingly, FC inhibited the expression of c-Myc, ribosomal protein L11 (L11) and nucleolin (NCL) in H1299 and H596 cells. Of note, silencing of L11 by siRNA transfection enhanced the expression of c-Myc through a negative feedback mechanism, while c-Myc knockdown downregulated L11 in H1299 cells. Additionally, combined treatment of FC and puromycin/doxorubicin promoted the activation of caspase 9/3, and attenuated the expression of c-Myc, Cyclin D1 and CDK4 in H1299 cells compared to single treatment. Taken together, our findings suggest that FC induces apoptosis and G1 arrest via regulation of ribosomal protein L11 and c-Myc and also enhances antitumor effect of puromycin or doxorubicin in NSCLCs.