Coptidis Rhizoma Extract Reverses 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells via Modulation of Thymidylate Synthase

Colorectal cancer (CRC) is a malignancy of the colon or rectum. It is ranked as the third most common cancer in both men and women worldwide. Early resection permitted by early detection is the best treatment, and chemotherapy is another main treatment, particularly for patients with advanced CRC. A well-known thymidylate synthase (TS) inhibitor, 5-fluorouracil (5-FU), is frequently prescribed to CRC patients; however, drug resistance is a critical limitation of its clinical application. Based on the hypothesis that Coptidis Rhizoma extract (CRE) can abolish this 5-FU resistance, we explored the efficacy and underlying mechanisms of CRE in 5-FU-resistant (HCT116/R) and parental HCT116 (HCT116/WT) cells. Compared to treatment with 5-FU alone, combination treatment with CRE and 5-FU drastically reduced the viability of HCT116/R cells. The cell cycle distribution assay showed significant induction of the G0/G1 phase arrest by co-treatment with CRE and 5-FU. In addition, the combination of CRE and 5-FU notably suppressed the activity of TS, which was overexpressed in HCT116/R cells, as compared to HCT116/WT cells. Our findings support the potential of CRE as an adjuvant agent against 5-FU-resistant colorectal cancers and indicate that the underlying mechanisms might involve inhibition of TS expression.     

Metformin Inhibits the Urea Cycle and Reduces Putrescine Generation in Colorectal Cancer Cell Lines

The urea cycle (UC) removes the excess nitrogen and ammonia generated by nitrogen-containing compound composites or protein breakdown in the human body. Research has shown that changes in UC enzymes are not only related to tumorigenesis and tumor development but also associated with poor survival in hepatocellular, breast, and colorectal cancers (CRC), etc. Cytoplasmic ornithine, the intermediate product of the urea cycle, is a specific substrate for ornithine decarboxylase (ODC, also known as ODC1) for the production of putrescine and is required for tumor growth. Polyamines (spermidine, spermine, and their precursor putrescine) play central roles in more than half of the steps of colorectal tumorigenesis. Given the close connection between polyamines and cancer, the regulation of polyamine metabolic pathways has attracted attention regarding the mechanisms of action of chemical drugs used to prevent CRC, as the drug most widely used for treating type 2 diabetes (T2D), metformin (Met) exhibits antitumor activity against a variety of cancer cells, with a vaguely defined mechanism. In addition, the influence of metformin on the UC and putrescine generation in colorectal cancer has remained unclear. In our study, we investigated the effect of metformin on the UC and putrescine generation of CRC in vivo and in vitro and elucidated the underlying mechanisms. In nude mice bearing HCT116 tumor xenografts, the administration of metformin inhibited tumor growth without affecting body weight. In addition, metformin treatment increased the expression of monophosphate (AMP)-activated protein kinase (AMPK) and p53 in both HCT116 xenografts and colorectal cancer cell lines and decreased the expression of the urea cycle enzymes, including carbamoyl phosphate synthase 1 (CPS1), arginase 1 (ARG1), ornithine trans-carbamylase (OTC), and ODC. The putrescine levels in both HCT116 xenografts and HCT116 cells decreased after metformin treatment. These results demonstrate that metformin inhibited CRC cell proliferation via activating AMPK/p53 and that there was an association between metformin, urea cycle inhibition and a reduction in putrescine generation.     

Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms

Polyphyllin Ι is a steroidal saponin isolated from the rhizoma of Paris polyphylla. In the present study, we aimed to investigate the anticancer effects of polyphyllin Ι in colorectal cancer and to elucidate the potential underlying molecular mechanisms. Using, CCK8 assay, flow cytometry, laser confocal microscope analysis and western blot, the anticancer effects of the polyphyllin Ι were analysed in colorectal cells. Our results indicate that polyphyllin Ι significantly decreased cell viability of HCT 116 cells and induced autophagy. Furthermore, we found that polyphyllin Ι induced autophagy in an ROS-dependent cell death and not related with PI3 K/AKT/mTOR pathway. We also provide evidence that excessive ROS triggered by polyphyllin Ι could induce G2/M phase arrest via regulating cycle proteins expression of cell cycle regulators, such as p21 and cyclinB1. In conclusion, polyphyllin Ι exhibit anticancer effect through ROS-dependent autophagy and induces G2/M arrest in colorectal cancer.     

Design,synthesis and biological evaluation of novel phthalazinone acridine derivatives as dual PARP and Topo inhibitors for potential anticancer agents

In this study,we designed and synthesized a series of phthalazinone acridine derivatives as dual PARP and Topo inhibitors.MTT assays indicated that most of the compounds significantly inhibited multiple cancer cells proliferation.In addition,all the compounds displayed Topo Ⅱ inhibition activity at 10 mol/L,and also possessed good PARP-1 inhibitory activities.Subsequent mechanistic studies showed that compound 9 a induced remarkable apoptosis and caused prominent S cell cycle arrest in HCT116 cells.Our study suggested that 9 a inhibiting Topo and PARP concurrently can be a potential lead compound for cancer therapy.     

A new curcumin derivative, HBC, interferes with the cell cycle progression of colon cancer cells via antagonization of the Ca2+/calmodulin function

HBC (4-[3,5-Bis-[2-(4-hydroxy-3-methoxy-phenyl)-ethyl]-4,5-dihydro-pyrazol-1-yl]-benzoic acid) is a recently developed curcumin derivative which exhibits potent inhibitory activities against the proliferation of several tumor cell lines. In the present study, we identified Ca2+/calmodulin (Ca2+/CaM) as a direct target protein of HBC using phage display biopanning. Ca2+/CaM-expressing phages specifically bound to the immobilized HBC, and the binding was Ca2+ dependent. Moreover, flexible docking modeling demonstrated that HBC is compatible with the binding cavity for a known inhibitor, W7, in the C-terminal hydrophobic pocket of Ca2+/CaM. In biological systems, HBC induced prolonged phosphorylation of ERK1/2 and activated p21(WAF1) expression, resulting in the induction of G0/G1 cell cycle arrest in HCT15 colon cancer cells. These results suggest that HBC inhibits the cell cycle progression of colon cancer cells via antagonizing of Ca2+/CaM functions.     

Inhibitory Effects and Mechanism of the Natural Compound Diaporthein B Extracted from Marine-Derived Fungi on Colon Cancer Cells

This study aimed to investigate the inhibitory effects and mechanism of diaporthein B (DTB), a natural compound extracted from the fungus Penicillium sclerotiorum GZU-XW03-2, on human colon cancer cells. The inhibitory effect of DTB at different concentrations on the proliferation of colon cancer cells HCT116 and LOVO was detected at 24 and 48 h. The effect of cell migration and clone formation ability were detected by cell scratch and plate cloning experiments. Morphological changes were observed by Hoechst 33342 and Annexin-V/PI staining, and flow cytometry was used to detect the proportion of apoptotic cells. DTB significantly inhibited colon cancer cell proliferation, migration, and apoptosis in a dose-dependent manner without significant effects on normal colonic epithelial cells NCM460. The IC50 inhibition effect can be achieved after treatment with 3 μmol/L DTB for 24 h. Compared with the blank group, the migration and clonal-forming ability of colon cancer cells in the DTB group was significantly decreased (p < 0.01), while the apoptotic cells were significantly increased (p < 0.01) in a concentration-dependent manner. DTB can inhibit the proliferation and migration of human colon cancer cells HCT116 and LOVO and promote the apoptosis of human colon cancer cells.     

The Effect of Different Ester Chain Modifications of Two Guaianolides for Inhibition of Colorectal Cancer Cell Growth

Several sesquiterpene lactones (STLs) have been tested as lead drugs in cancer clinical trials. Salograviolide-A (Sal-A) and salograviolide-B (Sal-B) are two STLs that have been isolated from Centaurea ainetensis, an indigenous medicinal plant of the Middle Eastern region. The parent compounds Sal-A and Sal-B were modified and successfully prepared into eight novel guaianolide-type STLs (compounds 1–8) bearing ester groups of different geometries. Sal-A, Sal-B, and compounds 1–8 were tested against a human colorectal cancer cell line model with differing p53 status; HCT116 with wild-type p53 and HCT116 p53^−/− null for p53, and the normal-like human colon mucosa cells with wild-type p53, NCM460. IC₅₀ values indicated that derivatization of Sal-A and Sal-B resulted in potentiation of HCT116 cell growth inhibition by 97% and 66%, respectively. The effects of the different molecules on cancer cell growth were independent of p53 status. Interestingly, the derivatization of Sal-A and Sal-B molecules enhanced their anti-growth properties versus 5-Fluorouracil (5-FU), which is the drug of choice in colorectal cancer. Structure-activity analysis revealed that the enhanced molecule potencies were mainly attributed to the position and number of the hydroxy groups, the lipophilicity, and the superiority of ester groups over hydroxy substituents in terms of their branching and chain lengths. The favorable cytotoxicity and selectivity of the potent molecules, to cancer cells versus their normal counterparts, pointed them out as promising leads for anti-cancer drug design.     

Induction of DR5-Dependent Apoptosis by PGA2 through ATF4-CHOP Pathway

Prostaglandin (PG) A₂, a cyclopentenone PG, induced apoptosis in both HCT116 and HCT116 p53 −/− cells. Although PGA₂-induced apoptosis in HCT116 cells was dependent on the p53-DR5 pathway, the mechanism underlying PGA₂-induced apoptosis in HCT116 p53 −/− cells remains unknown. In this study, we observed that PGA₂ caused an increase of mRNA expression of DR5 and protein expression even in HCT116 p53 −/− cells, accompanied by caspase-dependent apoptosis. Knockdown of DR5 expression by RNA interference inhibited PGA₂-induced apoptosis in HCT116 p53 −/− cells. Parallel to the induction of apoptosis, PGA₂ treatment upregulated expression of genes upstream of DR5 such as ATF4 and CHOP. Knockdown of CHOP prevented DR5-dependent cell death as well as the expression of DR5 protein. Furthermore, knockdown of ATF4 by RNA interference decreased both mRNA and protein levels of CHOP and DR5, thereby suppressing PGA₂-induced cell death. Consistently, the DR5 promoter activity increased by PGA₂ was not stimulated when the CHOP binding site in the DR5 promoter was mutated. These results collectively suggest that PGA₂ may induce DR5-dependent apoptosis via the ATF4-CHOP pathway in HCT116 p53 null cells.     

Differential modulation of zinc-stimulated p21(Cip/WAF1) and cyclin D1 induction by inhibition of PI3 kinase in HT-29 colorectal cancer cells

Activation of the extra cellular signal regulated kinase (ERK) pathway is involved in both proliferation and growth arrest of cells depending on intensity and duration of stimuli. In this study, we have elucidated differential regulation of the zinc-stimulated p21(CiP/WAF1) and cyclin D1 activation by inhibition of phosphoinositide 3-kinase (PI3K). In HT-29 colorectal cancer cells, the ERK activities were increased by zinc, which was accompanied by the induction of p21(Cip/WAF1) and cyclin D1. However, in the HT-29 cells pre-treated with PI3K inhibitor, LY294002, zinc induced further the p21(CiP/WAF) induction whereas abrogated cyclin D1 induction. In addition, the induction of p21(Cip/WAF1) expression completely inhibited the incorporation of bromodeoxyuridine (BrdU) into the nucleus, indicating that p21(CiP/WAF1) is an important indicator for ERK-dependent growth arrest. These studies suggest presence of an inter-related regulatory mechanism of cell proliferation by ERK and PI3K pathways.     

Antiproliferative Efficacy of N-(3-chloro-4-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine,DW-8, in Colon Cancer Cells Is Mediated by Intrinsic Apoptosis

A novel series of 4-anilinoquinazoline analogues, DW (1–10), were evaluated for anticancer efficacy in human breast cancer (BT-20) and human colorectal cancer (CRC) cell lines (HCT116, HT29, and SW620). The compound, DW-8, had the highest anticancer efficacy and selectivity in the colorectal cancer cell lines, HCT116, HT29, and SW620, with IC₅₀ values of 8.50 ± 2.53 µM, 5.80 ± 0.92 µM, and 6.15 ± 0.37 µM, respectively, compared to the non-cancerous colon cell line, CRL1459, with an IC₅₀ of 14.05 ± 0.37 µM. The selectivity index of DW-8 was >2-fold in colon cancer cells incubated with vehicle. We further determined the mechanisms of cell death induced by DW-8 in SW620 CRC cancer cells. DW-8 (10 and 30 µM) induced apoptosis by (1) producing cell cycle arrest at the G2 phase; (2) activating the intrinsic apoptotic pathway, as indicated by the activation of caspase-9 and the executioner caspases-3 and 7; (3) nuclear fragmentation and (4) increasing the levels of reactive oxygen species (ROS). Overall, our results suggest that DW-8 may represent a suitable lead for developing novel compounds to treat CRC.     

Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient’s survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study’s aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2^Y15, p-cdc2^T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21^Waf1/Cip1, the expression and association of p21^Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21^Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21^Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21^Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21^Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21^Waf1/Cip1-dependent signaling pathway in the NSCLC cells.     

Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer

Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations. Objectives: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA₂₀₂₀ and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study. Methods: The PamChip^® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells. Results: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA₂₀₂₀ with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA₂₀₂₀ with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG₁/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G₁ phase, p21/p27, and apoptosis in HT29-5FU cells. Conclusion: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA₂₀₂₀ and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.     

Pyrrolizine/Indolizine-NSAID Hybrids: Design,Synthesis, Biological Evaluation,and Molecular Docking Studies

In the current study, eight new hybrids of the NSAIDs, ibuprofen and ketoprofen with five pyrrolizine/indolizine derivatives were designed and synthesized. The chemical structures of these hybrids were confirmed by spectral and elemental analyses. The antiproliferative activities of these hybrids (5 μM) was investigated against MCF-7, A549, and HT-29 cancer cell lines using the cell viability assay, MTT assay. The results revealed 4–71% inhibition of the growth of the three cancer cell lines, where 8a,e,f were the most active. In addition, an investigation of the antiproliferative activity of 8a,e,f against MCF-7 cells revealed IC₅₀ values of 7.61, 1.07, and 3.16 μM, respectively. Cell cycle analysis of MCF-7 cells treated with the three hybrids at 5 μM revealed a pro-apoptotic increase in cells at preG1 and cell cycle arrest at the G1 and S phases. In addition, the three hybrids induced early apoptotic events in MCF-7 cells. The results of the molecular docking of the three hybrids into COX-1/2 revealed higher binding free energies than their parent compounds 5a,c and the co-crystallized ligands, ibuprofen and SC-558. The results also indicated higher binding free energies toward COX-2 over COX-1. Moreover, analysis of the binding modes of 8a,e,f into COX-2 revealed partial superposition with the co-crystallized ligand, SC-558 with the formation of essential hydrogen bonds, electrostatic, or hydrophobic interactions with the key amino acid His90 and Arg513. The new hybrids also showed drug-likeness scores in the range of 1.06–2.03 compared to ibuprofen (0.65) and ketoprofen (0.57). These results above indicated that compounds 8a,e,f deserve additional investigation as potential anticancer candidates.     

A Natural Degradant of Curcumin,Feruloylacetone Inhibits Cell Proliferation via Inducing Cell Cycle Arrest and a Mitochondrial Apoptotic Pathway in HCT116 Colon Cancer Cells

Feruloylacetone (FER) is a natural degradant of curcumin after heating, which structurally reserves some functional groups of curcumin. It is not as widely discussed as its original counterpart has been previously; and in this study, its anticancer efficacy is investigated. This study focuses on the suppressive effect of FER on colon cancer, as the efficacious effect of curcumin on this typical cancer type has been well evidenced. In addition, demethoxy-feruloylacetone (DFER) was applied to compare the effect that might be brought on by the structural differences of the methoxy group. It was revealed that both FER and DFER inhibited the proliferation of HCT116 cells, possibly via suppression of the phosphorylated mTOR/STAT3 pathway. Notably, FER could significantly repress both the STAT3 phosphorylation and protein levels. Furthermore, both samples showed capability of arresting HCT116 cells at the G2/M phase via the activation of p53/p21 and the upregulation of cyclin-B. In addition, ROS elevation and changes in mitochondrial membrane potential were revealed, as indicated by p-atm elevation. The apoptotic rate rose to 36.9 and 32.2% after being treated by FER and DFER, respectively. In summary, both compounds exhibited an anticancer effect, and FER showed a greater proapoptotic effect, possibly due to the presence of the methoxy group on the aromatic ring.     

Effects of Artonin E on Cell Growth Inhibition and Apoptosis Induction in Colon Cancer LoVo and HCT116 Cells

Today, colon cancer is the leading cause of cancer death. In Thailand, colon cancer is the third most common cancer in men and the second in women. Currently, the treatments for colon cancer include surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, targeted drug therapy, and stem cell therapy. However, some treatments have side effects for cancer patients, causing unwanted symptoms. In addition, targeted therapy comes with a high cost for patients. Therefore, bioactive compounds might be a good choice for colon cancer treatment. In this study, we investigated the effect of artonin E on apoptosis induction in colon cancer LoVo and HCT116 cells. The concentration ranges of artonin E at 3, 5, 10, and 30 µg/mL in LoVo cells and 1, 1.5, 2, and 3 µg/mL in HCT116 cells were examined. The results implied that artonin E decreased cell viability and increased apoptotic cells in a dose-dependent manner. In addition, artonin E stimulated mitochondrial membrane potential (ΔΨm) changes associated with apoptosis by increasing the sub-G1 population analyzed by flow cytometry. Western blotting showed that artonin E increased the proapoptotic protein, Bax, and decreased anti-apoptotic proteins’ (Bcl-2 and Bcl-x) expression. Moreover, artonin E also increased cleaved caspase-7 and cleaved-PARP expression in both LoVo and HCT116 cells. Interestingly, artonin E induced apoptosis through p-ERK1/2, p-p38/p38, and p-c-Jun expression in both cells. Our results suggested that artonin E induced apoptosis via caspase activation associated with the MAPKs signaling pathway. Therefore, artonin E might be used as a potential anticancer drug for colon cancer in the future.     

Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21(Waf1), p27(Kip1) and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21(Waf1) was increased, while p27(Kip1) and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21(Waf1) over-expression.     

p21Cip/WAF1 activation is an important factor for the ERK pathway dependent anti-proliferation of colorectal cancer cells

p21Cip/WAF1, an important regulator of cell proliferation, is induced by both p53- and extracellular signal regulated kinase (ERK) pathways. The induction of p21Cip/WAF1 occurs by prolonged activation of the ERKs caused by extracellular stimuli, such as zinc. However, not all the cells appeared to respond to ERK pathway dependent p21Cip/WAF1 induction. Here we investigated the cause of such difference using colorectal cancer cells. p21Cip/WAF1 induction and concomitant reduction of bromodeoxyuridine (BrdU) incorporation were observed by zinc treatment within HT-29 and DLD-1. However, HCT-116 cells with high endogenous p21Cip/WAF1 levels did not show any additional increment of p21Cip/WAF1 levels by zinc treatment and did maintain high BrdU incorporation level. The p21Cip/WAF1 induction by zinc depended upon prolonged activation of extracellular signal regulated kinase (ERK) was not observed in HCT-116 cells. The percentage of BrdU positive cells was 50% higher in p21Cip/WAF1 -/- HCT-116 cells compared to p21Cip/WAF1 +/+ HCT- 116 cells, and no cells induced p21Cip/WAF1 incorporated BrdU in its nucleus, yet confirming the importance of p21Cip/WAF1 induction in anti- proliferation. These results again support that p21Cip/WAF1 induction is a determinant in the regulation of colonic proliferation by the ERK pathway.     

Synthesis and Biological Evaluation of Harmirins,Novel Harmine–Coumarin Hybrids as Potential Anticancer Agents

As cancer remains one of the major health burdens worldwide, novel agents, due to the development of resistance, are needed. In this work, we designed and synthesized harmirins, which are hybrid compounds comprising harmine and coumarin scaffolds, evaluated their antiproliferative activity, and conducted cell localization and cell cycle analysis experiments. Harmirins were prepared from the corresponding alkynes and azides under mild reaction conditions using Cu(I) catalyzed azide–alkyne cycloaddition, leading to the formation of the 1H-1,2,3-triazole ring. Antiproliferative activity of harmirins was evaluated in vitro against four human cancer cell lines (MCF-7, HCT116, SW620, and HepG2) and one human non-cancer cell line (HEK293T). The most pronounced activities were exerted against MCF-7 and HCT116 cell lines (IC₅₀ in the single-digit micromolar range), while the most selective harmirins were 5b and 12b, substituted at C-3 and O-7 of the β-carboline core and bearing methyl substituent at position 6 of the coumarin ring (SIs > 7.2). Further experiments demonstrated that harmirin 12b is localized exclusively in the cytoplasm. In addition, it induced a strong G1 arrest and reduced the percentage of cells in the S phase, suggesting that it might exert its antiproliferative activity through inhibition of DNA synthesis, rather than DNA damage. In conclusion, harmirin 12b is a novel harmine and coumarin hybrid with significant antiproliferative activity and warrants further evaluation as a potential anticancer agent.