Abedul Haque

FLLL12 induces apoptosis in lung cancer cells through a p53/p73-independent but death receptor 5-dependent pathway

Unlike chemotherapy drugs, the safety of natural compounds such as curcumin has been well established. However, the potential use of curcumin in cancer has been compromised by its low bioavailability, limited tissue distribution and rapid biotransformation leading to low in vivo efficacy. To circumvent these problems, more potent and bioavailable analogs have been synthesized. In the current study, we investigated the mechanism of anti-tumor effect of one such analog, FLLL12, in lung cancers. IC50 values measured by sulforhodamine B (SRB) assay at 72 h and apoptosis assays (annexin V staining, cleavage of PARP and caspase-3) suggest that FLLL12 is 5-10-fold more potent than curcumin against a panel of premalignant and malignant lung cancer cell lines, depending on the cell line. Moreover, FLLL12 induced the expression of death receptor-5 (DR5). Ablation of the expression of the components of the extrinsic apoptotic pathway (DR5, caspase-8 and Bid) by siRNA significantly protected cells from FLLL12-induced apoptosis (p < 0.05). Analysis of mRNA expression revealed that FLLL-12 had no significant effect on the expression of DR5 mRNA expression. Interestingly, inhibition of global phosphatase activity as well as protein tyrosine phosphatases (PTPs), but not of alkaline phosphatases, strongly inhibited DR5 expression and significantly inhibited apoptosis (p < 0.05), suggesting the involvement of PTPs in the regulation of DR5 expression and apoptosis. We further showed that the apoptosis is independent of p53 and p73. Taken together, our results strongly suggest that FLLL12 induces apoptosis of lung cancer cell lines by posttranscriptional regulation of DR5 through activation of protein tyrosine phosphatase(s).

In vitro and in vivo synergistic antitumor activity of the combination of BKM120 and erlotinib in head and neck cancer: Mechanism of apoptosis and resistance.

We previously reported that the EGFR-targeted inhibitor erlotinib induces G1 arrest of squamous cell carcinoma of the head and neck (SCCHN) cell lines without inducing significant apoptosis. Large-scale genomic studies suggest that >50% of SCCHN cases have activation of PI3K pathways. This study investigated whether cotargeting of EGFR and PI3K has synergistic antitumor effects and apoptosis induction. We examined growth suppression, apoptosis, and signaling pathway modulation resulting from single and combined targeting of EGFR and PI3K with erlotinib and BKM120, respectively, in a panel of SCCHN cell lines and a xenograft model of SCCHN. In a panel of 12 cell lines, single targeting of EGFR with erlotinib or PI3K with BKM120 suppressed cellular growth without inducing significant apoptosis. Cotargeting of EGFR and PI3K synergistically inhibited SCCHN cell line and xenograft tumor growth, but induced variable apoptosis; some lines were highly sensitive, others were resistant. Mechanistic studies revealed that the combination inhibited both axes of the mTORC1 (S6 and 4EBP1) pathway in apoptosis-sensitive cell lines along with translational inhibition of Bcl-2, Bcl-xL, and Mcl-1, but failed to inhibit p-4EBP1, Bcl-2, Bcl-xL, and Mcl-1 in an apoptosis-resistant cell line. siRNA-mediated knockdown of eIF4E inhibited Bcl-2 and Mcl-1 and sensitized this cell line to apoptosis. Our results strongly suggest that cotargeting of EGFR and PI3K is synergistic and induces apoptosis of SCCHN cell lines by inhibiting both axes of the AKT–mTOR pathway and translational regulation of antiapoptotic Bcl-2 proteins. These findings may guide the development of clinical trials using this combination of agents. Mol Cancer Ther; 16(4); 729–38. ©2017 AACR.

Preclinical In Vitro, In Vivo, and Pharmacokinetic Evaluations of FLLL12 for the Prevention and Treatment of Head and Neck Cancers

Despite its high promise for cancer prevention and therapy, the potential utility of curcumin in cancer is compromised by its low bioavailability and weak potency. The purpose of the current study was to assess the in vitro and in vivo efficacy and pharmacokinetic parameters of the potent curcumin analogue FLLL12 in SCCHN and identify the mechanisms of its antitumor effect. IC50 values against a panel of one premalignant and eight malignant head and neck cancer cell lines as well as apoptosis assay results suggested that FLLL12 is 10- to 24-fold more potent than natural curcumin depending on the cell line and induces mitochondria-mediated apoptosis. In vivo efficacy (xenograft) and pharmacokinetic studies also suggested that FLLL12 is significantly more potent and has more favorable pharmacokinetic properties than curcumin. FLLL12 strongly inhibited the expression of p-EGFR, EGFR, p-AKT, AKT, Bcl-2, and Bid and increased the expression of Bim. Overexpression of constitutively active AKT or Bcl-2 or ablation of Bim or Bid significantly inhibited FLLL12-induced apoptosis. Further mechanistic studies revealed that FLLL12 regulated EGFR and AKT at transcriptional levels, whereas Bcl-2 was regulated at the translational level. Finally, FLLL12 strongly inhibited the AKT downstream targets mTOR and FOXO1a and 3a. Taken together, our results strongly suggest that FLLL12 is a potent curcumin analogue with more favorable pharmacokinetic properties that induces apoptosis of head and neck cancer cell lines by inhibition of survival proteins including EGFR, AKT, and Bcl-2 and increasing of the proapoptotic protein Bim. Cancer Prev Res; 9(1); 63–73. ©2015 AACR.

Inhibition of autotaxin alleviates inflammation and increases the expression of sodium-dependent glucose cotransporter 1 and Na+/H+ exchanger 3 in SAMP1/Fc mice

Crohns disease (CD) is a chronic, relapsing, inflammatory disease that is often associated with malnutrition because of inflammation in the small intestine. Autotaxin (ATX) is a secreted enzyme that produces extracellular lysophosphatidic acid. Increasing evidence suggests that ATX is upregulated during inflammation, and inhibition of ATX has been effective in attenuating chronic inflammatory conditions, such as arthritis and pulmonary fibrosis. This study aims to determine whether inhibition of ATX alleviates CD-associated inflammation and malnutrition by using SAMP1/Fc mice, a model of CD-like ileitis. SAMP1/Fc mice were treated the ATX inhibitor PF-8380 for 4 wk. Inhibition of ATX led to increased weight gain in SAMP1/Fc mice, decreased T helper 2 cytokine expression, including IL-4, IL-5, and IL-13, and attenuated immune cell migration. SAMP1/Fc mice have low expression of Na+-dependent glucose transporter 1 (SGLT1), suggesting impaired nutrient absorption associated with ileitis. PF-8380 treatment significantly enhanced SGLT1 expression in SAMP1/Fc mice, which could reflect the increased weight changes. However, IL-4 or IL-13 did not alter SGLT1 expression in Caco-2 cells, ruling out their direct effects on SGLT1 expression. Immunofluorescence analysis showed that the expression of sucrase-isomaltase, a marker for intestinal epithelial cell (IEC) differentiation, was decreased in inflamed regions of SAMP1/Fc mice, which was partially restored by PF-8380. Moreover, expression of Na+/H+ exchanger 3 was also improved by PF-8380, suggesting that suppression of inflammation by PF-8380 enhanced IEC differentiation. Our study therefore suggests that ATX is a potential target for treating intestinal inflammation and restoration of the absorptive function of the intestine. NEW & NOTEWORTHY This study is the first, to our knowledge, to determine whether autotoxin (ATX) inhibition improves inflammation and body weights in SAMP1/Fc mice, a mouse model of ileitis. ATX inhibition increased body weights of SAMP1/Fc mice and increased Na+-dependent glucose transporter 1 (SGLT1) expression. Increased SGLT1 expression in the inflamed regions was not a direct effect of cytokines but an indirect effect of increased epithelial cell differentiation upon ATX inhibition.

Abstract 4097: Src-met signaling confers apoptosis resistance to the combination of BKM120 and erlotinib in head and neck cancer

Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. However, intrinsic and acquired resistance to targeted agents poses challenges to the success of such treatments. The purpose of the current study was to investigate the intrinsic resistance of head and neck cancer (HNC) cell lines to apoptosis induced by the combination of EGFR inhibitor erlotinib and PI3K inhibitor BKM120. Methods: In a panel of 10 malignant and 1 premalignant HN cell lines, we evaluated cell growth inhibition (by SRB assay), IC50, combination index, dose reduction index (by CalcuSyn Software), and apoptosis (by Annexin-V staining). Protein expression levels were measured by Western blotting. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: Single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K demonstrated in vitro synergy in all except one cell line (based on combination index and dose reduction index) and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (Tu686, 686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, 1483, UMSSC90) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. JHU022 and 1483 cell lines expressed very high levels of p-cMet. Targeting cMet with the small molecule inhibitor crizotinib or siRNA rendered these cell lines highly sensitive to erlotinib and BKM120 induced apoptosis. Moreover, inhibition of Src family kinases with the small molecule inhibitor dasatinib or with c-Src-specific siRNA inhibited p-cMet. In addition, inhibition of SFK in these cell lines conferred sensitivity to erlotinib and BKM120 induced apoptosis. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K has synergistic activity, is effective in vivo, and induces apoptosis of some HNC cell lines. Two of the resistant cell lines express a high level of p-cMet as a downstream target of SFK, which confers resistance of these cells to the combination of erlotinib and BKM120. Citation Format: A.R.M. Ruhul Amin, Abu Anisuzzaman, Abedul Haque, Zhuo Chen, Dong Shin. Src-met signaling confers apoptosis resistance to the combination of BKM120 and erlotinib in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4097. doi:10.1158/1538-7445.AM2017-4097

Abstract 3502: Combination of BKM120 and erlotinib in squamous cell carcinoma of the head and neck: mechanism of in vitro and in vivo synergy

Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. The EGFR-targeted monoclonal antibody cetuximab is currently the only FDA-approved targeted treatment for head and neck cancers (HNC) with a response rate of less than 15%. Recent large scale genomic studies including TCGA suggest that >50% of HNC cases have activation of the PI3K/AKT/mTOR pathways, suggesting PI3K as an excellent target for HNC. The purpose of the current study is to investigate whether co-targeting EGFR and PI3K has synergistic antitumor effects and to understand the mechanism of synergy. Methods: In a panel of 10 malignant and 1 premalignant HN cell lines, we evaluated cell growth inhibition (by SRB assay); IC50, combination index, and dose reduction index (by CalcuSyn), and apoptosis (by Annexin-V staining). mRNA and protein expression were measured by qPCR and Western blotting, respectively. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: Single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K had in vitro synergy in all except one cell line (based on combination index and dose reduction index) and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (Tu686, 686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, 1483, UMSSC90) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. Only pan-PI3K inhibitors (BKM120 and BEZ235) induced effective apoptosis in combination with erlotinib, since the PI3K-α inhibitor BYL279 or allosteric mTORC1 inhibitor RAD001 failed to induce apoptosis in combination with erlotinib. Erlotinib strongly inhibited p-EGFR and p-ERK in vitro and in vivo, but only partially inhibited p-AKT. On the other hand, BKM120 completely inhibited p-AKT without affecting p-ERK. We also found that the combination of BKM120 and erlotinib strongly inhibited both axes of the AKT-mTORC1 pathway in sensitive cell lines, but failed to inhibit p-4EBP1 in one resistant cell line (UD-SCC2). In addition, the combination of BKM120 and erlotinib strongly inhibited Bcl-2, Bcl-xL and MCL-1 at the translational level in the sensitive cell lines but not the resistant one. siRNA-mediated knockdown of eIF4E (to inhibit 4EBP1-eIF4E-dependent translation) sensitized UD-SCC2 cells to the combination of erlotinib and BKM-induced apoptosis. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K is synergistic and induces apoptosis of HNC cell lines by inhibiting both axes of the AKT-mTOR pathway and translational regulation of anti-apoptotic Bcl-2 proteins. Citation Format: Abu Anisuzzaman, Abedul Haque, Zhuo Chen, Dong M. Shin, A.R.M. Ruhul Amin. Combination of BKM120 and erlotinib in squamous cell carcinoma of the head and neck: mechanism of in vitro and in vivo synergy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3502.

Abstract 29: Potent curcumin analog FLLL12 induces apoptosis in lung cancer cells through death receptor-5-dependent pathway

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Drug-associated toxicity is one of the major challenges in the management of cancer patients. Unlike chemotherapy drugs, the safety of natural compounds such as curcumin has been well established. However, the potential use of curcumin in cancer treatment has been compromised by its low bioavailability, limited tissue distribution, and rapid biotransformation leading to low efficacy. To circumvent these problems, more potent and bioavailable analogs have been synthesized. In the current study, we investigated the mechanism of anti-tumor effect of one such analog, FLLL12, in lung cancer cells. Methods: A panel of premalignant and malignant lung cancer cell lines was used for the study. SRB assay was used to measure cell growth inhibition and IC50. Annexin-V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: IC50 values (0.63-1.67 μM for FLLL12 as compared to 6.06-12.4 μM for curcumin, depending on the cell lines) and apoptosis results (annexin V staining and cleavage of PARP and caspase 3) suggest that FLLL12 is 5-10-fold more potent than curcumin against lung cancer cells. Moreover, FLLL12 induced the expression of death receptor-5 (DR5). Ablation of the expression of the components of the extrinsic apoptotic pathway (DR5, caspase 8 and BID) significantly protected cells from FLLL12-induced apoptosis as evidenced by reduced annexin V staining (p = 0.0008, p = 0.0001 and p = 0.0007 for DR5, caspase 8 and BID, respectively) and cleavage of PARP and caspase 3. Analysis of mRNA expression by RT-PCR revealed that FLLL-12 had no significant effect on the expression of DR5 mRNA. Interestingly, inhibition of global phosphatase activity by phosphatase inhibitor cocktail (PIC) completely abolished DR5 expression and significantly inhibited apoptosis (p = 0.0007 and p = 0.001, respectively) and the cleavage of casepase-3 and PARP. Similarly, inhibition of protein tyrosine phosphatases (PTPs) by sodium orthovanadate, but not by the alkaline phosphatase inhibitor imidazole, inhibited DR5 expression, apoptosis (p = 0.006) and cleavage of caspase-3 and PARP, suggesting the involvement of PTPs in the regulation of DR5 expression. FLLL12 also induced the expression of p53 and p73. However, inactivation of these proteins with their dominant negative construct or siRNA had no significant effects on apoptosis induction. Conclusions: Our results strongly suggest that FLLL12 induces apoptosis of lung cancer cell lines by posttranscriptional regulation of DR5 through activation of protein tyrosine phosphatase(s). This study was supported by NCI R03 CA171663, NCI P50 CA128613 and Robbins Scholar Award of Winship Cancer Institute of Emory University. Citation Format: Abedul Haque, Mohammad A. Rahman, James R. Fuchs, Zhuo G. Chen, Fadlo R. Khuri, Dong M. Shin, A.R.M. Ruhul Amin. Potent curcumin analog FLLL12 induces apoptosis in lung cancer cells through death receptor-5-dependent pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 29. doi:10.1158/1538-7445.AM2015-29

Abstract 2269: Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Effective chemopreventive strategies could save millions of lives. We have previously reported that the combination of the EGFR-TKI erlotinib and the green tea constituent epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. Methods: Well characterized and genetically validated squamous cell carcinoma of the head and neck cell lines were used throughout the study. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Gene overexpression and knockdown strategies were used to activate or shut down the expression of specific proteins. Results: siRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p=). On the other hand, overexpression of Bcl-2 significantly protected cells from apoptosis (p=), whereas overexpression of constitutively AKT had minimal effect on apoptosis (actual values). Analysis of mRNA expression by RT-PCR revealed that neither erlotinib, EGCG nor their combination had any significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2, suggesting post-transcriptional regulation of Bim, p21, p27 and Bcl-2 by the combination of erlotinib and EGCG. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim, and that inhibition of protein translation by cycloheximide pretreatment stabilized Bim, suggesting posttranslational regulation of Bim. Conclusion: Our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the post-translational level. Currently, a clinical trial is underway at Winship Cancer Institute of Emory University to inhibit or reverse the progression of oral premalignant lesions using this combination. (This study is supported by R03CA159369, P50CA128613 and Robbins Scholar Award of Winship Cancer Institute). Citation Format: Abedul Haque, Mohammad A. Rahman, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2269. doi:10.1158/1538-7445.AM2014-2269

Abstract 2283: Potent curcumin analog FLLL-12 targets both intrinsic and extrinsic signaling pathways to induce apoptosis in lung cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Curcumin is the major bioactive compound isolated from the rhizome of Curcuma longa (turmeric). Despite its high margin of safety and efficacy against various types of cancers, the potential utility of curcumin as a chemopreventive/chemotherapeutic drug is compromised by its low bioavailability and poor selectivity. To circumvent these problems, more potent and selective curcumin analogues have been developed. In the current study, we investigated the mechanism of apoptosis of one such synthetic analogue, FLLL-12, against lung cancers. This compound was reported to exhibit potent anti-tumor activity against prostate, colon and breast cancer cell lines. However, its mechanism of growth inhibition has yet to be elucidated. Methods: Multiple premalignant and fully transformed lung cancer cell lines were used throughout the study. SRB assay was used to measure cell growth inhibition. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Gene overexpression and knockdown strategies were used to activate or shut down of specific proteins. Results: IC50 values and apoptosis assay results showed that FLLL-12 was ∼10-fold more potent than the natural parent compound, curcumin, against lung cancer cells. Further mechanistic studies revealed that FLLL-12 induced the expression of DR5, inhibited the protein expression of EGFR, p-AKT, AKT and Bcl-2 and increased the expression of BIM. Analysis of mRNA expression suggested that FLLL-12 strongly inhibited the mRNA expression of EGFR and AKT, whereas the expression of Bcl-2 and Bim mRNAs remained unchanged. FLLL-12 also induced the expression of p53 and p73. However, inactivation of these proteins with their dominant negative construct or siRNA had no significant effects on apoptosis induction. Conclusions: Our results strongly suggest that FLLL-12 is a potent curcumin analog that induces apoptosis of lung cancer cell lines by targeting: (1) intrinsic pathways via transcriptional inhibition of EGFR and AKT and induction of BIM, and (2) extrinsic pathway via induction of DR5. Future in vivo studies using appropriate animal models are warranted for further development of this promising compound for cancer prevention and/or treatment for lung cancer. (Supported by R03CA171663, P50CA128613 and Robbins Scholar Award of Winship Cancer Institute of Emory University). Citation Format: A.R.M. Ruhul Amin, Abedul Haque, Mohammad A. Rahman, James R. Fuchs, Zhuo G. Chen, Dong M. Shin. Potent curcumin analog FLLL-12 targets both intrinsic and extrinsic signaling pathways to induce apoptosis in lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2014-2283