Targeted colorectal cancer treatment: In vitro anti-cancer effects of carnosine nanoparticles supported by agar and magnetic iron oxide.

The usage of peptides in the colorectal cancer (CRC) treatment promises to be a new anti-cancer therapy with improved treatment efficacy. Carnosine, a natural dipeptide molecule, has been demonstrated to be a potential anti-cancer drug. Nonetheless, it shows an exhibition of high-water solubility and is quickly degraded by carnosinase. Meanwhile, agar and magnetic iron oxide are the most used materials for drug delivery due to some of their advantages such as the low cost and the larger biocompatibility feature. The purpose of this study was to investigate the anti-cancer ability of agar-encapsulated carnosine nanoparticles (AgCa-NPs) and agar-encapsulated carnosine nanoparticles-coated magnetic iron oxide nanoparticles (AgCaN-MNPs) in human CRC cells, HCT-116. We evaluated the effects of AgCa-NPs and AgCaN-MNPs with a variety of concentrations (0, 5, 10, 15, 30, 40, or 50 mM) on HCT-116 cells after 72 h and 96 h by using MTT assay and observation cell morphology. We then analyzed the cell cycle progression and assessed the expression changes of genes related to apoptosis, autophagy, necroptosis, and angiogenesis after treatment for 96 h. The results showed that AgCa-NPs and AgCaN-MNPs in vitro study decreased HCT-116 cells viability. This effect was attributed to arrest of cell cycle, induction of programmed cell death, and suppression of angiogenesis by AgCa-NPs and AgCaN-MNPs. These findings revealed the antitumor efficacy of AgCa-NPs or AgCaN-MNPs for CRC treatment.

Glycated nisin enhances nisin's cytotoxic effects on breast cancer cells.

Antimicrobial peptides, such as nisin, are proposed as promising agents for cancer treatment. While glycation has been recognized as an effective method for enhancing various physicochemical properties of nisin, its anticancer effects remain unexplored. Therefore, we aimed to assess the anticancer potential of glycated nisin against MDA-MB-231 cells. The MDA-MB cells were treated with increasing concentrations of nisin and glycated nisin for 24, 48, and 72 h. The IC50 values for nisin were higher than those for glycated nisin. Glycated nisin at concentrations of 20 and 40 µg/mL decreased cell viability more than nisin at the same concentrations. The rate of apoptosis in the group treated with 20 µg/mL of nisin was lower compared to other treatment groups, and no significant difference in apoptosis rates was observed at different time points (p > 0.05). However, in the glycated nisin groups with concentrations of 10, 20, and 40 µg/mL, the level of apoptosis was very high after 24 h (73-81% of cells undergoing apoptosis). Overall, our study suggests that glycated nisin exhibits stronger cytotoxic effects on MDA-MB-231 cells, primarily involving the induction of apoptosis. This indicates its potential utilization as an alternative approach to address the issue of drug resistance in cancer cells.

"Top-down" overexpression optimization of butelase-1 in Escherichia coli and its application in anti-tumor peptides.

Butelase-1, the fastest known Asn/Asp-specific peptide ligase capable of catalyzing peptide ligation and cyclization, holds promising application prospects in the fields of food and biology. However, limited research exists on its recombinant expression and potential applications in peptide drugs. In this study, the activity of recombinantly-produced butelase-1 was enhanced by co-expressing it with a molecular chaperone in the SHuffle T7 strain. By introducing single or multiple synonymous rare codons at the beginning of the coding regions of beta-strand or alpha-helix, in combination with ribosomal binding site engineering, the activity of butelase-1 could be further improved. Consequently, the butelase-1 with a specific activity of 386.93 U/mg and a catalytic efficiency of 11,048 M-1 s-1 was successfully prepared in E. coli, resulting in a total activity of 8183.54 U/L and a yield of about 100 mg/L. This optimized butelase-1 was then used to efficiently cyclize the redesigned anti-cancer peptide lunasin, leading to enhanced bioavailability and anti-cancer effects. Overall, this study not only provided valuable biotechnology strategies for improving the recombinant expression of butelase-1 but also demonstrated a successful application for enhancing the biological efficacy of anti-cancer peptides.

Influences of Ipomoea batatas Anti-Cancer Peptide on Tomato Defense Genes.

AIMS: This study investigates the impact of IbACP (Ipomoea batatas anti-cancer peptide) on defense-related gene expression in tomato leaves, focusing on its role in plant defense mechanisms. BACKGROUND: Previously, IbACP was isolated from sweet potato leaves, and it was identified as a peptide capable of inducing an alkalinization response in tomato suspension culture media. Additionally, IbACP was found to regulate the proliferation of human pancreatic adenocarcinoma cells. OBJECTIVE: Elucidate IbACP's molecular influence on defense-related gene expression in tomato leaves using next-generation sequencing analysis. METHODS: To assess the impact of IbACP on defense-related gene expression, transcriptome data were analyzed, encompassing various functional categories such as photosynthesis, metabolic processes, and plant defense. Semi-quantitative reverse-transcription polymerase chain reaction analysis was employed to verify transcription levels of defense-related genes in tomato leaves treated with IbACP for durations ranging from 0 h (control) to 24 h. RESULTS: IbACP induced jasmonic acid-related genes (LoxD and AOS) at 2 h, with a significant up-regulation of salicylic acid-dependent gene NPR1 at 24 h. This suggested a temporal antagonistic effect between jasmonic acid and salicylic acid during the early hours of IbACP treatment. Downstream ethylene-responsive regulator genes (ACO1, ETR4, and ERF1) were consistently down-regulated by IbACP at all times. Additionally, IbACP significantly up-regulated the gene expressions of suberization-associated anionic peroxidases (TMP1 and TAP2) at all time points, indicating enhanced suberization of the plant cell wall to prevent pathogen invasion. CONCLUSION: IbACP enhances the synthesis of defense hormones and up-regulates downstream defense genes, improving the plant's resistance to biotic stresses.

Antimicrobial peptides CS-piscidin-induced cell death involves activation of RIPK1/PARP, and modification with myristic acid enhances its stability and tumor-targeting capability.

Ovarian cancer (OC) is a highly lethal gynecological malignancy, often diagnosed at advanced stages with limited treatment options. Here, we demonstrate that the antimicrobial peptide CS-piscidin significantly inhibits OC cell proliferation, colony formation, and induces cell death. Mechanistically, CS-piscidin causes cell necrosis by compromising the cell membrane. Furthermore, CS-piscidin can activate Receptor-interacting protein kinase 1 (RIPK1) and induce cell apoptosis by cleavage of PARP. To improve tumor targeting ability, we modified CS-piscidin by adding a short cyclic peptide, cyclo-RGDfk, to the C-terminus (CS-RGD) and a myristate to the N-terminus (Myr-CS-RGD). Our results show that while CS-RGD exhibits stronger anti-cancer activity than CS-piscidin, it also causes increased cytotoxicity. In contrast, Myr-CS-RGD significantly improves drug specificity by reducing CS-RGD toxicity in normal cells while retaining comparable antitumor activity by increasing peptide stability. In a syngeneic mouse tumor model, Myr-CS-RGD demonstrated superior anti-tumor activity compared to CS-piscidin and CS-RGD. Our findings suggest that CS-piscidin can suppress ovarian cancer via multiple cell death forms and that myristoylation modification is a promising strategy to enhance anti-cancer peptide performance.

In Silico Discovery of Anticancer Peptides from Sanghuang.

Anticancer peptide (ACP) is a short peptide with less than 50 amino acids that has been discovered in a variety of foods. It has been demonstrated that traditional Chinese medicine or food can help treat cancer in some cases, which suggests that ACP may be one of the therapeutic ingredients. Studies on the anti-cancer properties of Sanghuangporus sanghuang have concentrated on polysaccharides, flavonoids, triterpenoids, etc. The function of peptides has not received much attention. The purpose of this study is to use computer mining techniques to search for potential anticancer peptides from 62 proteins of Sanghuang. We used mACPpred to perform sequence scans after theoretical trypsin hydrolysis and discovered nine fragments with an anticancer probability of over 0.60. The study used AlphaFold 2 to perform structural modeling of the first three ACPs discovered, which had blast results from the Cancer PPD database. Using reverse docking technology, we found the target proteins and interacting residues of two ACPs with an unknown mechanism. Reverse docking results predicted the binding modes of the ACPs and their target protein. In addition, we determined the active part of ACPs by quantum chemical calculation. Our study provides a framework for the future discovery of functional peptides from foods. The ACPs discovered have the potential to be used as drugs in oncology clinical treatment after further research.

Isolation of Peptide Inhibiting SGC-7901 Cell Proliferation from Aspongopus chinensis Dallas.

Aspongopus chinensis Dallas is used as a traditional Chinese medicine as well as an edible insect. Although its anti-tumor effects have been observed, the anti-tumor active component(s) in the hemolymph of A. chinensis remains unknown. In this study, a combination usage of ultrafiltration, gel filtration chromatography, FPLC and RP-HPLC to separate and purify active peptides was performed based on the proliferation of the human gastric cancer SGC-7901 cell line treated with candidates. One peptide (MW = 2853.3 Da) was isolated from the hemolymph of A. chinensis. A total of 24 amino acid residues were continuously determined for the active peptide: N'-ECGYCAEKGIRCDDIHCCTGLKKK-C'. In conclusion, a peptide that can inhibit the proliferation of gastric cancer SGC-7901 cells in the hemolymph of A. chinensis was purified in this study, which is homologous to members of the spider toxin protein family. These results should facilitate further works for this peptide, such as the cloning of genes, expression in vitro by prokaryotic or eukaryotic systems, more specific tests of anti-tumor activity, and so on.

Anti-cancer Peptide Recognition Based on Grouped Sequence and Spatial Dimension Integrated Networks.

The diversification of the characteristic sequences of anti-cancer peptides has imposed difficulties on research. To effectively predict new anti-cancer peptides, this paper proposes a more suitable feature grouping sequence and spatial dimension-integrated network algorithm for anti-cancer peptide sequence prediction called GRCI-Net. The main process is as follows: First, we implemented the fusion reduction of binary structure features and K-mer sparse matrix features through principal component analysis and generated a set of new features; second, we constructed a new bidirectional long- and short-term memory network. We used traditional convolution and dilated convolution to acquire features in the spatial dimension using the memory network's grouping sequence model, which is designed to better handle the diversification of anti-cancer peptide feature sequences and to fully learn the contextual information between features. Finally, we achieved the fusion of grouping sequence features and spatial dimensional integration features through two sets of dense network layers, achieved the prediction of anti-cancer peptides through the sigmoid function, and verified the approach with two public datasets, ACP740 (accuracy reached 0.8230) and ACP240 (accuracy reached 0.8750). The following is a link to the model code and datasets mentioned in this article: https://github.com/ YouHongfeng101/ACP-DL.

Development of D-melittin polymeric nanoparticles for anti-cancer treatment.

Melittin, the primary peptide component of bee venom, is a potent cytolytic anti-cancer peptide with established anti-tumor activity. However, practical application of melittin in oncology is hampered by its strong, nonspecific hemolytic activity and intrinsic instability. To address these shortcomings, delivery systems are used to overcome the drawbacks of melittin and facilitate its safe delivery. Yet, a recent study revealed that encapsulated melittin remains immunogenic and can act as an adjuvant to elicit a fatal antibody immune response against the delivery carrier. We discovered that substitution of l-amino acids with d-amino acids mitigates this problem: D-melittin nanoformulations induce significantly decreased immune response, resulting in excellent safety without compromising cytolytic potential. We now report the first application of D-melittin and its micellar formulations for cancer treatment. D-melittin was delivered by a pH-sensitive polymer carrier that (i) forms micellar nanoparticles at normal physiological conditions, encapsulating melittin, and (ii) dissociates at endosomal pH, restoring melittin activity. D-melittin micelles (DMM) exhibits significant cytotoxicity and induces hemolysis in a pH-dependent manner. In addition, DMM induce immunogenic cell death, revealing its potential for cancer immunotherapy. Indeed, in vivo studies demonstrated the superior safety profile of DMM over free peptide and improved efficacy at prohibiting tumor growth. Overall, we present the first application of micellar D-melittin for cancer therapy. These findings establish a new strategy for safe, systemic delivery of melittin, unlocking a potential pathway toward clinical translation for cytotoxic peptides as anti-cancer agents. which can revolutionize in vivo delivery of therapeutic peptides and peptide antigens.

Development of a Cationic Amphiphilic Helical Peptidomimetic (B18L) As A Novel Anti-Cancer Drug Lead.

BST-2 is a novel driver of cancer progression whose expression confers oncogenic properties to breast cancer cells. As such, targeting BST-2 in tumors may be an effective therapeutic approach against breast cancer. Here, we sought to develop potent cytotoxic anti-cancer agent using the second-generation BST-2-based anti-adhesion peptide, B18, as backbone. To this end, we designed a series of five B18-derived peptidomimetics. Among these, B18L, a cationic amphiphilic α-helical peptidomimetic, was selected as the drug lead because it displayed superior anti-cancer activity against both drug-resistant and drug-sensitive cancer cells, with minimal toxicity on normal cells. Probing mechanism of action using molecular dynamics simulations, biochemical and membrane biophysics studies, we observed that B18L binds BST-2 and possesses membranolytic characteristics. Furthermore, molecular biology studies show that B18L dysregulates cancer signaling pathways resulting in decreased Src and Erk1/2 phosphorylation, increased expression of pro-apoptotic Bcl2 proteins, caspase 3 cleavage products, as well as processing of the caspase substrate, poly (ADP-ribose) polymerase-1 (PARP-1), to the characteristic apoptotic fragment. These data indicate that through the coordinated regulation of membrane, mitochondrial and signaling events, B18L executes cancer cell death and thus has the potential to be developed into a potent and selective anti-cancer compound.

In silico and In vitro Investigation of a Likely Pathway for Anti-Cancerous Effect of Thrombocidin-1 as a Novel Anticancer Peptide.

BACKGROUND: Antimicrobial and antifungal activities of Thrombocidin-1 (TC-1) is shown previously, however,.the anti-cancerous feature of this peptide is still uncovered. OBJECTIVE: The objective is to evaluate anti-cancerous feature of recombinant TC-1. METHODS: In this study, based on the significant similarity of rTC-1 and IL-8 in case of coding sequence, tertiary structure, and also docking and molecular dynamic simulation (MD) results with CXCR1, a receptor which has positive correlation with different cancers, a likely pathway for anticancerous effect of rTC-1 was proposed. In addition, the coding sequence of TC-1+6xhistidine (rTC-1) was inserted into the pET22b(+) vector and cloned and expressed by E. coli BL21 and finally purified through nickel affinity column. Afterward, the retrieved rTC-1 was used in MTT assay against mouse colon adenocarcinoma, hepatocellular carcinoma, chondrosarcoma, mouse melanoma, and breast adenocarcinoma cell lines to investigate its probable anticancer application. RESULTS: Docking and MD simulation results showed that rTC-1 and IL-8 share almost the same residues in the interaction with CXCR1 receptor. Besides, the stability of the rTC-1_CXCR11-38 complex was shown during 100ns MD simulation. In addition, the successful expression and purification of rTC-1 depict an 8kD peptide. The IC50 results of MTT assay revealed that rTC-1 has cytotoxic effect on C26-A and SW1353 cancerous cell lines. CONCLUSION: Therefore, apart from probable anti-cancerous effect of rTC-1 on C26-A and SW1353 cell lines, this peptide may be able to mimic the anti-cancerous pathway of IL-8.

Design and development of high affinity dual anticancer peptide-inhibitors against p53-MDM2/X interaction.

AIMS: Inhibition of P53-MDM2/X interaction is known as an effective cancer therapy strategy. In this regard, pDI peptide was introduced previously with the potential of targeting MDM2. In this research, the large-scale peptide mutation screening was used to achieve the best sequence of pDI with the highest affinity for inhibition activity against MDM2/X. MAIN METHODS: Three mutant peptides of pDI as dual inhibitor peptides including single mutations of pDIm/4W, pDIm/11M and double mutations of pDIdm/4W11M were presented with the high affinities to inhibit both MDM2/X. The selected mutants were then evaluated comprehensively to confirm their ability as potent MDM2/X inhibitors, using a theoretical simulation approach. KEY FINDINGS: MD simulations analyses confirmed their dual inhibition potential against both MDM2/X interactions with p53 protein. The developed pDIm and mainly pDIdm peptides showed stable conformations over the simulation time with conserved secondary structure and effective interaction with MDM2/X by physical binding such as hydrogen bonding. Besides, umbrella sampling free energy calculation indicated higher binding energy, ΔGbinding, of pDIm-MDM2/X and pDIdm-MDM2/X compared to pDI-MDM2/X. SIGNIFICANCE: The optimized and improved mutant pDI, pDIdm, with more effective ΔGbinding values of -30 and -25 kcal/mol to MDMX and MDM2, respectively, is recommended as a promising anticancer agent and suitable candidate for experimental evaluations.

Comparison of patient-derived high and low phosphatidylserine-exposing colorectal carcinoma cells in their interaction with anti-cancer peptides.

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi-)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti-cancer therapeutics. Peptide NK-2, derived from porcine NK-lysin, was originally discovered due to its broad-spectrum antimicrobial activities. Today, also potent anti-cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non-abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK-2 and structurally improved anti-cancer variants thereof against two patient-derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle-based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface-exposed phosphatidylserine is of crucial importance for the activity of peptide NK-2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Mastoparan is a membranolytic anti-cancer peptide that works synergistically with gemcitabine in a mouse model of mammary carcinoma.

Anti-cancer peptides (ACPs) are small cationic and hydrophobic peptides that are more toxic to cancer cells than normal cells. ACPs kill cancer cells by causing irreparable membrane damage and cell lysis, or by inducing apoptosis. Direct-acting ACPs do not bind to a unique receptor, but are rather attracted to several different molecules on the surface of cancer cells. Here we report that an amidated wasp venom peptide, Mastoparan, exhibited potent anti-cancer activities toward leukemia (IC50~8-9.2µM), myeloma (IC50~11µM), and breast cancer cells (IC50~20-24µM), including multidrug resistant and slow growing cancer cells. Importantly, the potency and mechanism of cancer cell killing was related to the amidation of the C-terminal carboxyl group. Mastoparan was less toxic to normal cells than it was to cancer cells (e.g., IC50 to PBMC=48µM). Mastoparan killed cancer cells by a lytic mechanism. Moreover, Mastoparan enhanced etoposide-induced cell death in vitro. Our data also suggest that Mastoparan and gemcitabine work synergistically in a mouse model of mammary carcinoma. Collectively, these data demonstrate that Mastoparan is a broad-spectrum, direct-acting ACP that warrants additional study as a new therapeutic agent for the treatment of various cancers.

Effect of nisin and doxorubicin on DMBA-induced skin carcinogenesis--a possible adjunct therapy.

In view of the emergence of multidrug-resistant cancer cells, there is a need for therapeutic alternatives. Keeping this in mind, the present study was aimed at evaluating the synergism between nisin (an antimicrobial peptide) and doxorubicin (DOX) against DMBA-induced skin carcinogenesis. The possible tumoricidal activity of the combination was evaluated in terms of animal bioassay observations, changes in hisotological architecture of skin tissues, in situ apoptosis assay (TUNEL assay) and in terms of oxidant and antioxidant status of the skin tissues. In vivo additive effect of the combination was evidenced by larger decreases in mean tumour burden and tumour volume in mice treated with the combination than those treated with the drugs alone. Histological observations indicated that nisin-DOX therapy causes chromatin condensation and marginalisation of nuclear material in skin tissues of treated mice which correlated well with the results of TUNEL assay wherein a marked increase in the rate of apoptosis was revealed in tissues treated with the combination. A slightly increased oxidative stress in response to the adjunct therapy as compared to dox-alone-treated group was revealed by levels of lipid peroxidation (LPO) and nitrite generation in skin tissue-treated mice. An almost similar marginal enhancement in superoxide dismutase levels corresponding with a decrease in catalase activity could also be observed in nisin + DOX-treated groups as compared to nisin and dox-alone-treated groups. These results point towards the possible use of nisin as an adjunct to doxorubicin may help in developing alternate strategies to combat currently developing drug resistance in cancer cells.

Entry of a cationic lytic-type peptide into the cytoplasm via endocytosis-dependent and -independent pathways in human glioma U251 cells.

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.

Localization of the anti-cancer peptide EGFR-lytic hybrid peptide in human pancreatic cancer BxPC-3 cells by immunocytochemistry.

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti-cancer epithelial growth factor receptor (EGFR)-lytic hybrid peptide, a 32-amino-acid peptide composed of an EGFR-binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR-lytic hybrid peptide in BxPC-3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR-lytic peptide compared with non-treated cells. In confocal laser scanning microscopy, immunostaining of EGFR-lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR-lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR-lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR-lytic peptide; however, no effect on cell death after EGFR-lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR-lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death.

IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis.

A 16-amino-acid peptide was isolated from the leaves of sweet potato. The peptide caused a rapid alkalinization response in tomato suspension culture media, a characteristic of defense peptides in plants. No post-translational modification was observed on the peptide according to MALDI-MS analysis. We have named the peptide Ipomoea batatas anti-cancer peptide (IbACP). IbACP also was shown with the ability to dose-dependently inhibit Panc-1, a pancreatic cancer line, cell proliferation. The morphological observations of the Panc-1 cells by phase contrast microscopy showed significant changes after treatment with IbACP. Moreover, caspase-3 and PARP [poly(ADP-ribose) polymerase] were activated by IbACP treatment, followed by cell death. An increase in the levels of cleaved caspase-3 and -9 was also detected by an immunoblot assay after treatment with IbACP. In addition, genomic DNA fragmentation and decreased cellular proliferation were induced when IbACP was supplied to the Panc-1 cells, further demonstrating its biological relevance. The combined data indicates that IbACP peptide may have an important role in the regulation of cellular proliferation by inducing and promoting apoptosis through the mitochondrial apoptotic pathway. This report also showed that IbACP peptide contains potent anti-cancer effects and may play an important role in herbal medicine development.