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PreprintCited by 10Cell death mechanisms and regulationAntimicrobial Peptides and ActivitiesChemical Synthesis and Analysis

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Chemokine-derived oncolytic peptide induces immunogenic cancer cell death and significantly suppresses tumor growth

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Furukawa, Natsuki, Yang, Wendy, Chao, Alex, Patil, Akash, Mirando, Adam, Pandey, Niranjan, Popel, Aleksander·2023·Cell Death Discovery
DOI 10.21203/rs.3.rs-3335225/v1PMC PMC10987543

Preprint — published as 10.1038/s41420-024-01932-5 · Paper page

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Abstract

Chemokinostatin-1 (CKS1) is a 24-mer peptide originally discovered as an anti-angiogenic peptide derived from the CXCL1 chemokine. Here, we demonstrate that CKS1 acts not only as an anti-angiogenic peptide but also as an oncolytic peptide due to its structural and physical properties. CKS1 induced both necrotic and apoptotic cell death specifically in cancer cells while showing minimal toxicity in non-cancerous cells. Mechanistically, CKS1 disrupted the cell membrane of cancer cells quickly after treatment and activated the apoptotic pathway at later time points. Furthermore, immunogenic molecules were released from CKS1 treated cells, indicating that CKS1 induces immunogenic cell death. CKS1 effectively suppressed tumor growth in vivo. Collectively, these data demonstrate that CKS1 is a unique peptide that functions both as an anti-angiogenic peptide and as an oncolytic peptide and has a therapeutic potential to treat cancer.

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Fig. 1 The structure and physical properties of CKS1. A The structure of CKS1 predicted by AlphaFold 2. B The electrostatic potential of the surface of CKS1 was predicted by the APBS electrostatics plugin in Pymol. Blue regions are the positively charged regions and red regions are the negatively charged regions. C The hydrophobic regions of CKS1 are visualized as red and the hydrophilic regions are visualized as white.
Fig. 1 The structure and physical properties of CKS1. A The structure of CKS1 predicted by AlphaFold 2. B The electrostatic potential of the surface of CKS1 was predicted by the APBS electrostatics plugin in Pymol. Blue regions are the positively charged regions and red regions are the negatively charged regions. C The hydrophobic regions of CKS1 are visualized as red and the hydrophilic regions are visualized as white.CC BY · PubMed Central
Fig. 2 CKS1 induces rapid cell death in multiple cancer cell lines but not in non-cancerous cell lines. A Cell viability of 4T1 cells after CKS1 treatment was measured by a real-time cell analyzer (RTCA). The vertical, dotted line indicates the time of peptide addition, and solid lines and the shaded areas show the mean and the standard error of the technical replicates, respectively. Representative of N = 3. B–E The cell viability and the release of LDH were measured after the cells were treated with the indicated concentration of CKS1 for 6 h. The blue data points and the red data points show the cell viability and the release of LDH, respectively. The release of LDH in the supernatant was measured by the CyQUANT LDH cytotoxicity assay and was normalized to the amount of LDH released from cells treated with lysis buffer. The curves show the dose-response curves fitted to the observed data. Each data point shows the technical replicate of each experiment. Representative of N = 3 independent experiments.
Fig. 2 CKS1 induces rapid cell death in multiple cancer cell lines but not in non-cancerous cell lines. A Cell viability of 4T1 cells after CKS1 treatment was measured by a real-time cell analyzer (RTCA). The vertical, dotted line indicates the time of peptide addition, and solid lines and the shaded areas show the mean and the standard error of the technical replicates, respectively. Representative of N = 3. B–E The cell viability and the release of LDH were measured after the cells were treated with the indicated concentration of CKS1 for 6 h. The blue data points and the red data points show the cell viability and the release of LDH, respectively. The release of LDH in the supernatant was measured by the CyQUANT LDH cytotoxicity assay and was normalized to the amount of LDH released from cells treated with lysis buffer. The curves show the dose-response curves fitted to the observed data. Each data point shows the technical replicate of each experiment. Representative of N = 3 independent experiments.CC BY · PubMed Central
Fig. 3 CKS1 rapidly disrupts the cell membrane of cancer cells. A 4T1 cells were treated with water or 100 µM CKS1 for 30 min and the detailed structure of the cell was observed using transmission electron microscopy. The cell membrane of 4T1 cells was disrupted after 30 min of CKS1-treatment. B 4T1 cells were treated with 100 µM FITC-CKS1 (green) and monitored by fluorescence live cell imaging. The timestamp indicates the minutes after cells were treated with the peptide. Representative of N = 3. C 4T1 cells were stained with calcein-AM (green) and were treated with 100 µM CKS1. The timestamp indicates the minutes after cells were treated with the peptides. D The ratio of cells maintaining calcein in the cytosol at each time point was calculated based on the live cell imaging. Representative of N = 3.
Fig. 3 CKS1 rapidly disrupts the cell membrane of cancer cells. A 4T1 cells were treated with water or 100 µM CKS1 for 30 min and the detailed structure of the cell was observed using transmission electron microscopy. The cell membrane of 4T1 cells was disrupted after 30 min of CKS1-treatment. B 4T1 cells were treated with 100 µM FITC-CKS1 (green) and monitored by fluorescence live cell imaging. The timestamp indicates the minutes after cells were treated with the peptide. Representative of N = 3. C 4T1 cells were stained with calcein-AM (green) and were treated with 100 µM CKS1. The timestamp indicates the minutes after cells were treated with the peptides. D The ratio of cells maintaining calcein in the cytosol at each time point was calculated based on the live cell imaging. Representative of N = 3.CC BY · PubMed Central
Fig. 4 CKS1 diminishes mitochondrial membrane potential and activates the apoptotic pathway. A 4T1 cells were treated with water or 100 µM CKS1 for 6 h and the detailed structure of the cell was observed using transmission electron microscopy. Overall shrinkage of the cell, collapse of the nucleus, chromatin condensation (arrow), and apoptotic blebbing (triangle) indicate that the cells underwent apoptosis. B Fluorescence live cell imaging of 4T1 cells stained with JC-1 and treated with 100 µM CKS1. The timestamp indicates the minutes after CKS1 treatment (negative value indicates pre-treatment). Green indicates the monomer form of JC-1, and red indicates the polymer form of JC-1. C Twenty-five cells were randomly selected from the video of figure A (supplemental video 5) and the ratio between the green intensity and the red intensity was calculated. Means ± SEM, representative of N = 3 independent experiments, Dunnett’s test (*p < 0.05, ***p < 0.005). D At least ten fields of view were acquired for each time point and the fraction of cells with red intensity in the nucleus (cells with active caspase activity) was calculated. Means ± SEM, representative of N = 3 independent experiments, Dunnett’s test (*p < 0.05, ***p < 0.005).
Fig. 4 CKS1 diminishes mitochondrial membrane potential and activates the apoptotic pathway. A 4T1 cells were treated with water or 100 µM CKS1 for 6 h and the detailed structure of the cell was observed using transmission electron microscopy. Overall shrinkage of the cell, collapse of the nucleus, chromatin condensation (arrow), and apoptotic blebbing (triangle) indicate that the cells underwent apoptosis. B Fluorescence live cell imaging of 4T1 cells stained with JC-1 and treated with 100 µM CKS1. The timestamp indicates the minutes after CKS1 treatment (negative value indicates pre-treatment). Green indicates the monomer form of JC-1, and red indicates the polymer form of JC-1. C Twenty-five cells were randomly selected from the video of figure A (supplemental video 5) and the ratio between the green intensity and the red intensity was calculated. Means ± SEM, representative of N = 3 independent experiments, Dunnett’s test (*p < 0.05, ***p < 0.005). D At least ten fields of view were acquired for each time point and the fraction of cells with red intensity in the nucleus (cells with active caspase activity) was calculated. Means ± SEM, representative of N = 3 independent experiments, Dunnett’s test (*p < 0.05, ***p < 0.005).CC BY · PubMed Central
Fig. 5 The cationic helical structure of CKS1 is important for its activity. A The structure of CKS1, NC1, and NC2. B, C The release of LDH in the supernatant was measured after treating 4T1 cells with 100 µM of the indicated peptides for 6 h. Data were normalized to cells treated with lysis buffer. Means ± SEM, N = 3, Dunnett’s test (***p < 0.005).
Fig. 5 The cationic helical structure of CKS1 is important for its activity. A The structure of CKS1, NC1, and NC2. B, C The release of LDH in the supernatant was measured after treating 4T1 cells with 100 µM of the indicated peptides for 6 h. Data were normalized to cells treated with lysis buffer. Means ± SEM, N = 3, Dunnett’s test (***p < 0.005).CC BY · PubMed Central
Fig. 6 CKS1 induces the release of immunogenic molecules. A, B Cells were treated with 20 µM or 100 µM CKS1 for 30 min and the amount of ATP released in the culture media was quantified. Means ± SEM, N = 3, Dunnet’s test (*p < 0.05, ***p < 0.005). C, D Cells were treated with 100 µM CKS1 for 6 h and the amount of HMGB-1 released in the culture media was quantified. Means ± SEM, N = 3, Welch’s t-test (*p < 0.05). E DC2.4 cells were treated with either complete media (control), conditioned media from 4T1 cells, 100 µM CKS1, or conditioned media from 4T1 cells treated with 100 µM CKS1. The relative expression level normalized to the expression level in the control condition is shown. Means ± SEM, N = 4, Dunnet’s test (*p < 0.05, **p < 0.01).
Fig. 6 CKS1 induces the release of immunogenic molecules. A, B Cells were treated with 20 µM or 100 µM CKS1 for 30 min and the amount of ATP released in the culture media was quantified. Means ± SEM, N = 3, Dunnet’s test (*p < 0.05, ***p < 0.005). C, D Cells were treated with 100 µM CKS1 for 6 h and the amount of HMGB-1 released in the culture media was quantified. Means ± SEM, N = 3, Welch’s t-test (*p < 0.05). E DC2.4 cells were treated with either complete media (control), conditioned media from 4T1 cells, 100 µM CKS1, or conditioned media from 4T1 cells treated with 100 µM CKS1. The relative expression level normalized to the expression level in the control condition is shown. Means ± SEM, N = 4, Dunnet’s test (*p < 0.05, **p < 0.01).CC BY · PubMed Central
Fig. 7 CKS1 induces necrosis and suppresses tumor growth in vivo. A CKS1 was injected intratumorally in established 4T1 tumors. After 24 h, tumors were excised and were stained with H&E stain. The white area indicates necrosis caused by CKS1. Representative of N = 3. B, C The tumor volume of 4T1 tumors and CT26 tumors were recorded. Fourty milligram/kg CKS1 was applied daily starting on day 7. Means ± SEM, 4T1 control N = 15, 4T1 CKS1 treatment N = 15, CT26 control N = 10, CT26 CKS1 treatment N = 10. Two-way repeated-measures ANOVA was performed to determine the significance of CKS1 treatment (***p < 0.005).
Fig. 7 CKS1 induces necrosis and suppresses tumor growth in vivo. A CKS1 was injected intratumorally in established 4T1 tumors. After 24 h, tumors were excised and were stained with H&E stain. The white area indicates necrosis caused by CKS1. Representative of N = 3. B, C The tumor volume of 4T1 tumors and CT26 tumors were recorded. Fourty milligram/kg CKS1 was applied daily starting on day 7. Means ± SEM, 4T1 control N = 15, 4T1 CKS1 treatment N = 15, CT26 control N = 10, CT26 CKS1 treatment N = 10. Two-way repeated-measures ANOVA was performed to determine the significance of CKS1 treatment (***p < 0.005).CC BY · PubMed Central

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Cites

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Metadata

Authors
Furukawa, N., Yang, W., Chao, A., Patil, A., Mirando, A., Pandey, N., & Popel, A.
Affiliations
Johns Hopkins University (US)
Corresponding author
Furukawa, Natsuki
ORCID
Venue
Cell Death Discovery
Publisher
Springer Science and Business Media LLC
Published
2023-10-03
Volume
10
Issue
1
Pages
161-161
ISSN
2058-7716 (ISSN-L)
DOI
10.21203/rs.3.rs-3335225/v1
PMC
PMC10987543
OpenAlex
W4393396983
Cited by
10
References
1
Citations by year
2026: 4 · 2025: 4 · 2024: 2
FWCI
1.36
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death Discovery (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health and Human Services (P30CA006973, R01 CA138264, S10OD016374)
  • Johns Hopkins University (P30CA006973)
  • National Institutes of Health (1s10od016374-01, CA006973)
  • National Cancer Institute (R01CA138264, P30CA006973)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
  • Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
Keywords
oncolytic peptide, immunogenic cell death, anti-angiogenic peptides, apoptosis, necrosis, cell membrane damage, tumor growth inhibition, cancer therapy

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