Our project aims to design and produce an ACP which can have combination effects with cisplatin against Non-Small Cell Lung Cancer (NSCLC). We have total designed fifteen BioBrick™ to express different anticancer peptides in our expression system. AC-P19 is an anticancer peptide which has been experimentally proved that it has anticancer ability against A549 cell line [BBa_5056009]. [1] We used it as a positive control. Four peptides from Cordyceps militaris, a well-known dietary therapy in anticancer treatment, were selected to test their effectiveness to lung cancer [BBa_5056005]-[BBa_5056008]. Also, we modified those five peptides (AC-P19, C-ori, C-rds, CTP-ori and CTP-rds) by adding linker and cell-penetrating peptide to increase anticancer ability via promoting cell-penetrating ability [BBa_5056011]-[BBa_5056015]. Furthermore, we used RFdiffusion and ProteinMPNN-AI to generate all-new peptide sequences by targeting PDEδ, which regulates KRAS signaling, a common gene mutation in cancerous cells. Linker and cell-penetrating peptide are also added at end of those de novo peptides to increase its cell-penetrating ability [BBa_5056000]-[BBa_5056004].
SacI and NdeI were used to cut open the pET plasmid, Cordyceps militaris ACPs and AC-P19 and then both insert and fragments were ligated. The diagrams below show that Cordyceps militaris ACPs and AC-P19 carried by the plasmid provided by IDT company are successfully digested by SacI and NdeI. For each insert, target bands are observed, although the each target bands are not clear, as inserts are too short (< 100bp). Below show the results.
SacI and NdeI were used to cut open the pET plasmid and also five de novo peptides (ACP1, ACP5, ACP7, ACP11 and ACP12), four Cordyceps militaris ACP with cell-penetrating peptide(CPP) and AC-P19 with cell-penetrating peptide and then both insert and fragments were ligated. The diagrams below show that all inserts carried by the plasmid provided by IDT company were successfully digested by SacI and NdeI. Target bands are observed, so all target genes were cut successfully. Below show the results.
Total 14 recombinant plasmids were transformed into BL21 and colonies were observed in LB plate with kanamycin. Colony PCR were carried out and then AC-P19, C-ori, C-rds, ACP1, ACP5 and C-rds-CPP get +ve results in some colonies. Below shows the results:
The constructed expression vector of AC-P19, C-ori, C-rds, ACP1, ACP5 and C-rds-CPP was inserted into BL21 for expression. The protein was induced with 0.4mM IPTG for 24hrs at 20℃. And then, the cells were harvested by centrifugation at 6000xg for 20mins. Samples were collected and would run SDS-PAGE later. 1 ml Lysis buffer with 1mM PMSF per 10mL culture was used to resuspend the pellet. Suspension was sonicated on ice (Amplitude: 95%; Time 5 min; Pulse 2 son, 2 s off). It was cleared by centrifugation at 150000 rpm, 4℃ for 15 mins.
Suspension of above peptides (AC-P19, C-ori, C-rds, ACP1, ACP5 and C-rds-CPP) were purified by Ni-NTA resin and eluted in PBS. We repeated this step three times. And then, SDS-PAGE has been conducted to confirm whether purification succeeds.
Protein band at about 3-6kDa were observed in elution for AC-P19, C-ori, C-rds, ACP1, ACP5 and C-rds-CPP before and after purification. The concentration of AC-P19, C-ori, C-rds, ACP1, ACP5 and C-rds-CPP were further measured by nanodrop.
To increase the concentration of the peptide. We used the Amicon Ultra-0.5 mL Centrifugal Filters for protein purification and concentration. Below table shows the concentration of each peptide before and after concentration.
The A549 cancer cells showed certain degree of retarded growth under the inverted microscope after incubating with medium containing 50μM AC-P19 for two days . It is our pioneer study, showing that anti-cancer peptide is worthy of further investigationp
Cisplatin does not have significant effect on the cell viability of the A549 cancer cells with concentrations between 1.56μM and 3.13μM. However, cell viability showed an apparent decline when the concentration of cisplatin continues to increase.
The IC50 value is estimated 5.33μM [2], which is quite similar to 6.14μM, as determined by some cisplatin related research on cell line A549[3]. It shows that the cell line used is not particularly sensitive or resistant to cancer drug.
The effects of two peptide drugs, C-ori and C-rds, on cell viability at different concentrations (0, 25 µM, and 50 µM) was investigated (Figure 23). Both peptide drugs C-ori and C-rds did not exhibit significant cytotoxic effects on the cells at the tested concentrations (25 µM and 50 µM). The lack of cytotoxicity also implies that they may not be effective as anticancer agents in this context, as they did not reduce cell viability significantly, even at high concentration as 50 µM.
The experimental data showed that the ACPs from Cordyceps militaris (C-ori, C-rds, C-rds-CPP) are less effective at killing A549 compared with ACP1 and ACP5 (KAPI). At concentration of 50µM, all peptide drug, except ACP5 (KAPI), has no significant effects compared to the control. The anti-cancer abilities of ACP5 (KAPI) is much higher than the remaining peptide durgs. At concentration of 50µM, the percentage of cell viability inhibition for A549 with ACP5 (KAPI) is approximately 70%. This shows that ACP5 (KAPI) is the more competent in anti-cancer ability compared with other ACPs.
The dosage response of A549 to KAPI was assessed (n=2). According to the results, KAPI has little effect on the cell viability of the A549 cancer cells with concentrations between 1.56μM and 6.25μM. However, the cell viability showed an apparent decline when the concentration was increased to 12.5μM, 25μM and 50μM with cell viability of approximately 70%, 55% and 18% respectively. The IC50 of KAPI is determined to be 25.9μM.
The cytotoxicity assay was conducted with the peptide drug ACP5 (ACP5 (KAPI)) on cell line BEAS-2B (n=3). BEAS-2B, which is a non-cancerous bronchial epithelial cell line, served as a control to investigate the effect of the peptide drugs on non-cancerous human cells[4]. According to the results (Figure 26), it has been shown that ACP5 (KAPI) has a limited effect on the growth of the BEAS-2B cell line. This finding suggests that ACP5 (KAPI) does not alter the proliferation as well as viability of A549 on a large scale. The limited effect on BEAS-2B cells indicates that ACP5 (KAPI) may have a favorable safety profile, as it does not adversely affect normal healthy cells. This characteristic is crucial for the peptide drug, as it implies a lower likelihood of cytotoxicity in non-target tissues, thereby reducing potential side effects in clinical applications.
The combination effect of ACP5 (KAPI) and Cisplatin is studied. The result is shown in Figure 6. As indicated, as 25μM or 50μM of ACP5 (KAPI) is introduced, the percentage inhibition of cell viability is enhanced. In addition, it is shown that 12.5μM of cisplatin with 0μM ACP5 (KAPI) show similar degree of inhibition as 6.25μM with 25μM of ACP5 (KAPI). Hence, it is demonstrated that ACP5 (KAPI) has a good potential in reducing the required dosage of cisplatin in chemotherapy.
In 2D culture, cell viability decreases as the concentration of cisplatin increases, indicating that higher concentrations of the substance reduce cell viability. A similar trend is observed in 3D culture, with cell viability decreasing as the concentration increases. As the concentration increases, the difference in cell viability between 2D and 3D conditions becomes more pronounced, with 3D cultures generally showing slightly higher viability than 2D cultures at the same concentrations. It suggests that the 3D culture condition is more resistant to the effect of cisplatin compared to the 2D condition.
In assessing the effectiveness of ACP5 (KAPI) on tumour viability, it is shown that tumour viability is reduced by 20% after two days of drug treatment. The effect of longer treatment is yet to be studied with limited time.
To understand the impact of KAPI on key genes involved in the KRAS pathway, quantitative PCR (qPCR) was conducted to measure the expression levels of SOS1, EGFR, PI3K, and BRAF in A549 cells treated with 50uM KAPI, compared to untreated cells. (Figure 30)
Two upstream and two downstream markers of KRAS was chosen for the study. The result can be summarized as follows (Figure 31 and 32):
Fold Change:0.972
Nature: Upstream of KRAS in signaling pathway
Relationship with KRAS: SOS1 facilitates the exchange of GDP for GTP on RAS proteins, converting them from an inactive to an active state. This activation is essential for transmitting signals from cell surface receptors to the nucleus, promoting cell proliferation and survival [5]
Interpretation: The expression level of SOS1 remains relatively unchanged (fold change close to 1), indicating that KAPI treatment does not significantly affect SOS1 mRNA levels. This suggests that SOS1 may not be a primary target of KAPI or that its regulation is not directly influenced by PDE delta inhibition.
Fold Change: 1.522
Nature: Upstream of KRAS in signaling pathway
Relationship with KRAS: When the receptor EGFR receives extracellular growth factors such as EGF, it transmits signals to activate KRAS.
Interpretation: EGFR expression is upregulated by approximately 52% in response to KAPI treatment. This increase could be a compensatory mechanism where cells attempt to maintain signaling through the EGFR pathway despite the inhibition of KRAS signaling. EGFR upregulation might also indicate feedback activation as a response to disrupted downstream signaling in KRAS[5].
Fold Change: 0.632
Nature: Downstream of KRAS in signaling pathway
Relationship with KRAS: KRAS transmits signal and stimulate PI3K-AKT-mTOR pathway, which promotes cell survival and growth.
Interpretation: PI3K expression is reduced by approximately 37% with KAPI treatment. This downregulation suggests that KAPI effectively inhibits the PI3K pathway, which is a critical downstream effector of KRAS signaling. Reduced PI3K levels indicate a successful disruption of the signaling cascade, potentially leading to decreased cell proliferation and survival [2].
Fold Change: 0.115
Nature: Downstream of KRAS in signaling pathway
Relationship with KRAS: BRAF is activated by upstream signals from KRAS. Once activated, BRAF phosphorylates and activates MEK1 and MEK2, which then activate ERK1 and ERK2 in the MARK/ERK pathway, which controls cell proliferation.
Interpretation: BRAF expression is significantly downregulated (approximately 89% reduction) in response to KAPI treatment. This substantial decrease indicates that BRAF, another key downstream effector of KRAS, is highly sensitive to PDE delta inhibition by KAPI. The marked reduction in BRAF levels suggests a strong inhibitory effect on the MAPK/ERK pathway, which is crucial for cell growth and survival [3].
According to our hypothesis, KAPI targets PDEδto inhibit KRAS signalling in cancer cells. The qPCR results demonstrate that KAPI effectively modulates the expression of key genes in the KRAS signaling pathway. The significant downregulation of PI3K and BRAF, along with the upregulation of EGFR, highlights the complex regulatory mechanisms and potential compensatory responses in cancer cells. These findings support the potential of KAPI as a therapeutic agent targeting PDE delta to inhibit KRAS-driven oncogenic signaling.
In our wet lab experiments, the cytotoxic effect of KAPI on A549 is demonstrated. In qPCR, we studied the drug’s effect on specific pathways related to KRAS. Evidence shows that KRAS activity is inhibited under the effect of KAPI. However, this does not directly show whether the cell death is caused solely by the inhibition of KRAS. In order to demonstrate KRAS and PDEδ interaction under the effect of KAPI, confocal microscopy can be used to visualize and compare the co-localization of KAPI and PDEδ using fluorescently tagged versions of both molecules in different concentration of KAPI[6].
Here is the progress of our unfinished work and future plan. We are currently already working on this test. The plasmid containing the KRAS’s DNA sequence and EGFP, the green fluorescent protein is designed. (Figure 33)
Similarly, a plasmid containing the PDEδ‘s DNA sequence and another fluorescent protein, mCherry is designed
The plasmid construct is purchased from BGI Tech Solutions. The KRAS and PDE-delta were amplified using the following primers:
After PCR, Gel electrophoresis is carried out to ensure that the target KRAS and PDE-delta are amplified properly.
The amplified KRAS and PDEδ sequence were inserted into the GFP containing plasmid and mCherry containing plasmid respectively using SalI and NotI restriction sites following standard molecular cloning procedures. Both plasmids can then be transfected to A549. With the expression of fluorescently tagged versions of both molecules, the interaction between KRAS and PDEδ can be visualized and the degree of co-localization of KRAS and PDEδ can be compared with different concentration of KAPI [6].
Apart from the 4 genes targeted in previous experiments (SOS, EGFR, P13K, BRAF), further investigation can be conducted to determine the specific actions of KAPI on KRAS upstream and downstream pathways. For instance, common KRAS downstream pathways and effectors also include RAL-GEF and STAT3 , while upstream pathways include GAB 2 and SHP 1 and 2 etc.[7][8] They are all worth investigating to study the loss of KRAS function.
Western blot analysis can be used to measure protein expression in a sample, as well as its abundance and size. In order to further examine the KAPI’s impact on the cancer signal pathway, protein expression of important marker can be studied[9].
Combining KAPI with other inhibitor targeting cancer signal pathway is one of our future direction. Synergistic effect may be achieved to enhance efficiency and reduce chance of chemotherapy resistance. For example, SOS1 inhibitors with KRAS inhibitors improved the depth and durability of response in lung and colorectal cancer models. Restoration of response was observed in preclinical models rendered resistant to KRAS inhibitors. These results highlight the potential of SOS1 inhibitors to broaden the response to KRAS inhibitors in the clinic.[10]
[1] Int J Mol Sci. 2022 Dec; 23(24): 15594. Published online 2022 Dec 9. doi: 10.3390/ijms232415594
[2] The Quest Graph™ IC50 Calculator.https://www.aatbio.com/tools/ic50-calculator
[3] Tang Y, Hou J, Li G, Song Z, Li X, Yang C, Liu W, Hu Y, Xu Y. ABCG2 regulates the pattern of self-renewing divisions in cisplatin-resistant non-small cell lung cancer cell lines. Oncol Rep. 2014 Nov;32(5):2168-74. doi: 10.3892/or.2014.3470. Epub 2014 Sep 9. PMID: 25200103.
[4] M. Biola-Clier, D. Beal, S. Caillat, S. Libert, L. Armand, N. Herlin-Boime, S. Sauvaigo, T. Douki, M. Carriere, Comparison of the DNA damage response in BEAS-2B and A549 cells exposed to titanium dioxide nanoparticles, Mutagenesis, Volume 32, Issue 1, 1 January 2017, Pages 161
[5] Uribe, M. L., Marrocco, I., & Yarden, Y. (2021). EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired ResistanceCancers, 13(11), 2748. 2: Yang, J., Nie, J., Ma, X., Wei, Y., Peng, Y., & Wei, X. (2019). Targeting PI3K in cancer: mechanisms and advances in clinical trialsMolecular Cancer, 18, 26. 3: Poulikakos, P. I., Sullivan, R. J., & Yaeger, R. (2022). Molecular Pathways and Mechanisms of BRAF in Cancer Therapy. Clinical Cancer Research, 28(21), 4618-4628.
[6] Pawley, J. B. (Ed.). (2006). Confocal microscopy: Principles and applications for the study of cellular interactions. Springer.
[7] Zhu Z, Golay HG, Barbie DA. Targeting pathways downstream of KRAS in lung adenocarcinoma. Pharmacogenomics. 2014 Aug;15(11):1507-18. doi: 10.2217/pgs.14.108. PMID: 25303301; PMCID: PMC4227881.
[8] Saliani M, Jalal R, Ahmadian MR. From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers. Cancer Biol Med. 2019 Aug;16(3):435-461. doi: 10.20892/j.issn.2095-3941.2018.0530. PMID: 31565476; PMCID: PMC6743616.
[9] Osborne C, Brooks SA. SDS-PAGE and Western blotting to detect proteins and glycoproteins of interest in breast cancer research. Methods Mol Med. 2006;120:217-29. doi: 10.1385/1-59259-969-9:217. PMID: 16491604.
[10] SOS1 inhibitor combinations overcome KRAS inhibitor resistance. Nat Cancer. 2024 Sep;5(9):1294-1295. doi: 10.1038/s43018-024-00801-5. PMID: 39134714.