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Renieramycin T Derivative DH_22 Induces p53-dependent Apoptosis in Lung Cancer Cells

INDIANA GITA ANGGRAENI, ZIN ZIN EI, DAIKI HOTTA, MASASHI YOKOYA and PITHI CHANVORACHOTE
In Vivo September 2023, 37 (5) 1960-1966; DOI: https://doi.org/10.21873/invivo.13292

Abstract

Background/Aim: Targeting apoptotic pathways has been identified as a promising strategy for the treatment of lung cancer. We synthesized a new derivative of renieramycin T (RT), named DH_22, and examined its anticancer activities in human lung cancer cells. Materials and Methods: The RT derivative DH_22 was chemically modified from RT. The apoptosis-inducing effect was evaluated in A549 cells by annexin V-FITC/PI staining and nuclear staining assay (Hoechst/PI). In addition, the molecular pathway was analyzed by western blot analysis. Results: In the cell viability and nuclear staining tests, DH_22 was discovered to be cytotoxic with an IC50 of 13.27 μM; it induced apoptosis of lung cancer cells. Regarding the mechanism, DH_22 contributed to the activation of p53-dependent apoptosis and decreased the cellular level of c-Myc. The p53-dependent mechanism was indicated by an increase in p53, an induction of the pro-apoptotic Bax protein, and a decrease in the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein. Conclusion: DH_22 has great potential for further development as a new anticancer drug.

Key Words:

Lung cancer cases have a high mortality rate, accounting for 2 million diagnoses and 1.8 million deaths worldwide (1). Molecular abnormalities are clearly associated with lung cancer development. One notable characteristic of cancer is the ability of tumor cells to evade apoptosis (2). Apoptosis, or programmed cell death, is triggered by intracellular signals. This process is crucial for cell homeostasis and elimination of cancer cells (3). Targeting apoptosis pathways has emerged as a promising strategy for cancer therapy. p53 is one of the genes that can initiate the apoptosis program. In response to DNA-damaging stimuli, p53 induces apoptosis or cell cycle arrest. p53 is the most comprehensively characterized tumor suppressor. This gene is frequently mutated in malignancies, inactivating the proapoptotic function of p53 and contributing to the drug-resistant phenotype (4).

p53 initiates responses such as cell cycle arrest, apoptosis, DNA repair, and cell differentiation by activating the transcription of specific target genes containing p53 DNA binding sites. Furthermore, the Bcl-2 protein family is one of the primary regulators of the mitochondria-mediated pathway to apoptosis. The mechanism of apoptosis may depend on p53, which stimulates the expression of several Bcl-2 family genes, including Bax. p53 induces Bax expression, shifting the balance between pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins. Bcl-2 inhibits apoptosis by blocking Bax homodimerization and preventing mitochondrial outer membrane permeabilization (5) to release cytochrome c, activating the caspase cascade, and resulting in apoptotic cell death (6).

Marine organisms offer biologically active compounds with the potential for new drug discovery (7). Renieramycin T (RT) is a tetrahydroisoquinoline alkaloid molecule isolated from the blue sponge Xestospongia sp. that has been pretreated with potassium cyanide (8). Its anti-cancer properties have recently been examined in colon (HCT116), prostate (DU145) (9), non-small cell lung (H292, H460, and QG56) (10), breast (T47D), and pancreatic (AsPC1) cancer cells (8). However, despite the remarkable anti-cancer effects, the complex structure of RT makes large scale synthesis difficult. Accordingly, a simpler RT derivative, DH_22 compound, was synthesized and its anti-cancer activity was determined.

The discovery of DH_22 was primarily based on the presence of pyridine. The nucleus of pyridines is a well-studied six-membered heterocyclic moiety with a wide range of biological and medicinal applications (11, 12). With its established anti-cancer property, pyridine is an impactful and valued moiety in the field of pharmaceutical sciences. Several prominent anti-cancer medicines reportedly include a pyridine component (13). In addition, an essential function of pyridine in medicinal chemistry is to increase water solubility due to its low basicity and catalytic property (14, 15). This study aimed to investigate the anticancer activity of the RT derivative DH_22 through the intrinsic apoptosis pathway, which is mediated by p53-dependent apoptosis.

Materials and Methods

Preparation of the DH_22 stock solution. The compound was dissolved in dimethyl sulfoxide (DMSO) to form a 50 mM stock solution and stored at −20°C. It was freshly diluted to the concentrations used in the experiments with the awareness that the final concentration of DMSO should be less than 0.05%.

Cell lines and culture. A549 (ATCC® CCL-185TM, RRID: CVCL 0014) human non-small cell lung cancer (NSCLC) cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The A549 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM). The medium was kept in a humidified incubator at 37°C with 5% CO2, supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/ml of penicillin and streptomycin.

Cytotoxicity assay. The A549 lung cancer cells (1×104 cells/well) were seeded in a 96-well plate and incubated overnight. The next day, the cells were treated with 0–100 mM of the renieramycin T derivative DH-22 for 24 h at 37°C, followed by the addition of 100 ml (4 mg/ml in PBS) 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) solution (Sigma Chemical, St. Louis, MO, USA). The plate was kept in the incubator for four h at 37°C. After that, the MTT reagent was discarded, and 100 ml DMSO was added to solubilize the formazan crystals. A microplate reader (Anthros, Durham, NC, USA) was used to measure the formazan product at 570 nm. The half maximal inhibitory concentration (IC50) in different groups was calculated using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

Annexin V-FITC/PI staining apoptotic assay. A549 cells were seeded (5×104 cells/well) and treated for 16 h with varying concentrations of DH-22 (0-10 mM). Then, cells were centrifuged, washed twice with PBS, and suspended in binding buffer. The cells were then stained for 15 min at room temperature with 2.5 ml of Annexin V-FITC and 1 ml of PI according to the manufacturer’s protocol (ImmunoTools, Friesoythe, Germany). The Guava easyCyte flow cytometer was used to examine live, apoptotic, and necrotic cells (EMD Millipore, Hayward, CA, USA).

Nuclear staining assay (Hoechst33342/PI). Hoechst 33342 and PI double staining methods were used for nuclear co-staining to differentiate between apoptotic and necrotic cell death. In a 96-well plate, A549 cells were seeded at a density of 1×104 cells/well. The plate was incubated overnight to allow the cells to adhere. After that, cells were incubated with a 0–50 mM concentration of renieramycin T derivative (DH_22) for 16 h. Then, the cells were stained with 10 mg/ml of Hoechst 33342 (Sigma) and 0.02 mg/ml of propidium iodide (PI) (Sigma) for 30 min. A fluorescent microscope was used to visualize the cell. The percentage of apoptotic cells was calculated by counting the number of condensed nuclear and DNA-fragmented cells.

Western blot analysis. The western blot method was used to measure the levels of specific proteins in cells. A549 cells were seeded in six-well plates at a density of 4x105 cells/well overnight. DH-22, was then applied to the cells for 16 h at a concentration of 0-20 mM. The protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Samples were transferred to 0.2 mm polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA), and probed overnight at 4°C with the specific primary antibodies. A chemiluminescence substrate was used to generate a signal that was recorded using an X-ray film. The ImageJ program was used to assess protein band intensity (version 1.52, National Institutes of Health, Bethesda, MD, USA).

Statistical analysis. The results are presented as means±SEM of at least three biologically replicated samples. Statistical differences were analyzed using one-way ANOVA analysis followed by a post hoc test in Graph Pad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA). Statistical significance is determined at p<0.05.

Results

Cytotoxicity and apoptosis-inducing effect of DH-22. The right-half structure of renieramycin T, which was discovered in the Thai blue sponge Xestopongia sp., served as the starting material for the synthesis of DH-22. The step-by-step synthesis of DH_22 is shown in Figure 1. This study examined the cytotoxic profile of DH-22 in non-small cell lung cancer (NSCLC) A549 cells to discover its possible anti-cancer effects. The cells were incubated with DH-22 (0–100 μM) for 24 h. The findings show that DH-22 decreased the viability of A549 cells considerably (Figure 2A), with half maximum inhibitory concentration (IC50) of DH-22 of 13.27±0.66 μM. Flow cytometric analysis of apoptosis and necrosis using annexin V-FITC/PI staining was conducted to confirm the type of cell death (Figure 2B and C). A549 cells were treated for 16 h with DH-22 (0-10 mM), and the findings revealed that the cytotoxic effect is due to apoptotic cell death.

Figure 1.
Figure 1.

Synthesis of the renieramycin T derivative DH-22.

Figure 2.
Figure 2.

Effect of DH-22 on cell viability and apoptotic cell death in the NSCLC cell line A549. (A) A significant decrease in cell viability after 24 h exposure to DH-22 is observed with the half maximum inhibitory concentration (IC50) of 13.27±0.66 μM. (B) After 16 h of treatment with DH22, apoptotic and necrotic A549 cells were determined using Annexin V-FITC/PI staining and flow cytometry. (C) Percentage of each cell death-type as determined using flow cytometry. All data are presented as means±SEM (n=3). ***p<0.001 compared with untreated cells.

DH-22 induces apoptosis cell death. Nuclear morphological alteration has been widely used as an apoptosis indicator. Microscopy demonstrates nuclear condensation, cell contraction, and cell fragmentation into apoptotic bodies. The nuclear morphology of DH-22-treated cells was analyzed using Hoechst 33342. A549 cells were treated with 0–50 μM DH-22 for 16 h. Propidium iodide (PI) was also utilized to identify necrotic cell death. Exposure to DH-22 (0–50 μM) resulted in apoptotic cell death, which was clearly indicated by the detection of DNA condensation and/or fragmentation (Figure 3A and B). Similarly, treatment with the increasing concentrations of DH-22 resulted in decreased cell viability and increased percentage of apoptotic cells.

Figure 3.
Figure 3.

Apoptotic characteristics of renieramycin T derivative DH_22 treated cells. (A) After A549 cells were treated with DH-22 for 16 h, Hoechst 33342/PI staining was used to stain the cell nuclei. (B) The percentage of apoptotic cells as determined by Hoechst/PI staining. (C) Treatment with-20 μM DH-22 for 16 h induces cleavage of PARP and Caspase-9. (D) The average relative density. The data is presented as mean±SEM (n=3). *p<0.05, ***p<0.001 compared to untreated control cells.

Furthermore, a novel approach to studying the presence of apoptosis is to demonstrate the activation of downstream caspases. This can be achieved using western blotting of target proteins cleaved by caspases, or DNA repair enzymes. Increased levels of the apoptotic marker proteins cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP) were found in treated cells (Figure 3C and D). Caspase-9 is an initiator caspase that is activated by the intrinsic apoptotic pathway and cleaves effector caspases, ultimately resulting in apoptosis. During apoptosis, PARP is cleaved by caspases, resulting in the loss of its enzymatic activity and the accumulation of PARP fragments.

Mechanism of action of DH-22 in induction of apoptosis. The main regulators of p53-dependent apoptosis, such as p53, anti-apoptotic protein (Bcl-2), and pro-apoptotic protein (Bax), were investigated in A549 cells treated with DH-22 (0-20 μM) for 16 h. Bax is a well-known direct target of the p53 tumor suppressor protein. The results demonstrated that p53 and Bax were significantly elevated in response to the DH-22 treatment. However, the levels of the anti-apoptotic protein Bcl-2 were reduced (Figure 4). The reduction of Bcl-2 is a crucial step in the early stages of apoptosis. It permits caspases and other pro-apoptotic factors to be activated, resulting in cell death. These findings demonstrate that DH-22 induces apoptosis in A549 cells via stimulating p53-dependent pathways and altering critical regulatory proteins.

Figure 4.
Figure 4.

The results of western blot analysis of several proteins. (A) The levels of apoptosis-associated proteins p53, Bax, and Bcl-2 in A549 cells treated with DH-22 (0-20 μM) for 16 h. (B) The average relative density. The data is presented as mean±SEM (n=3). *p<0.05, ***p<0.001 compared to untreated control cells.

Discussion

The RT derivative DH-22 (Figure 1) is a novel compound synthesized based on the right-half structure of RT, or RM-based hybrid renieramycin-ecteinascidin analog (8), which was isolated from the Thai blue sponge Xestopongia sp. We synthesized various derivatives of renieramycin from marine natural products as starting materials (16) and evaluated their biological activities. The results indicated that a compound was effective when aromatic rings (especially pyridine rings) were introduced in place of the angelate in the C-1 substituent of renieramycins (17, 18). The substituent at N-3 of the tricyclic skeleton corresponds to the right half of renieramycin and is expected to affect the C-1 side chain of renieramycins. Thus, the pyridin-4-ylmethyl group of DH-22 was introduced with the expectation of acting as a pyridine ring of the C-1 side chain of the renieramycin analogue.

Our study found that DH-22 has a cytotoxic effect on human A549 cells with an IC50 value of 13.27±0.66 μM (Figure 2A). Subsequently, Annexin V/PI staining indicated that the elimination of cancer cells occurred through apoptosis (Figure 2B and C). Annexin V/PI staining, used for detecting both apoptotic and necrotic cells, relies on the ability of Annexin V to bind to phosphatidylserine, which is normally located on the inner leaflet of the plasma membrane but is exposed on the outer leaflet during apoptosis (19, 20). Annexin V-positive and PI-negative cells are considered to be in early-stage apoptosis, while Annexin V-positive and PI-positive cells are considered to be in late-stage apoptosis (21).

Additionally, morphological characteristics of apoptosis-related cytotoxicity were observed in cells treated with DH-22 (Figure 3A and B). Apoptotic cells typically appear bright with condensed nuclei and fragmented or condensed chromatin when stained with Hoechst. In contrast, necrotic cells appear swollen with irregularly shaped nuclei and dispersed chromatin (22, 23). Necrotic cells may also have cytoplasmic vacuolation or ruptured plasma membranes (24).

Apoptosis is the outcome of a cascade of molecular processes resulting in caspase activation and substrate cleavage. Caspases are cysteine proteases involved in the beginning and end of apoptosis. Caspase-9 is an initiator caspase that is activated in response to pro-apoptotic signals by the release of cytochrome c from the mitochondria. Caspase-9, once activated, cleaves and activates downstream effector caspases, such as caspase-3, resulting in the destruction of cellular components and cell death (25).

PARP, a nuclear enzyme involved in DNA repair, is one of the essential substrates of effector caspases. PARP cleavage by active caspases results in the loss of its enzymatic activity and the buildup of PARP cleavage fragments, which indicates that apoptosis is still occurring. The biochemical activities caused by caspase activation and PARP cleavage are also in line with the morphological alterations observed during apoptosis (Figure 3C and D).

The p53-dependent pathway, a key apoptotic pathway, was found to be activated by DH-22 treatment. The tumor suppressor protein p53 is involved in DNA repair, cell cycle arrest, and apoptotic cell death. It is activated in response to DNA damage by ataxia telangiectasia-mutated kinases (26). The activation of p53 alters the cellular balance of Bcl-2 family proteins, increasing the levels pro-apoptotic protein (Bax) while decreasing those of anti-apoptotic protein (Bcl-2) (Figure 4). The concurrent apoptotic effect was mediated via intrinsic mode through the up-regulation of Bax and activated cleaved caspase-9, as well as the down-regulation of Bcl-2 and full-length caspase-9 expression (27). The death-survival threshold is then modified by the competitive dimerization of pro- and anti-apoptotic proteins. The pro-apoptotic dimers may induce the release of mitochondrial contents into the cytoplasm, which activate caspase-9 (28). This process leads to the activation of PARP to avoid the depletion of energy (NAD and ATP), which is necessary for the later stages of apoptosis (29).

These findings indicate that DH-22 is a promising candidate for the development of novel anticancer drugs. Activation of the p53-dependent pathway and subsequent induction of apoptosis indicate that DH-22 has potential as a cancer treatment agent. Further research is required to determine the molecular mechanisms underlying the apoptotic effects of DH-22.

Conclusion

In summary, DH_22 is a novel compound synthesized from marine natural products that has been shown to efficiently induce apoptosis in cancer cells. DH-22 treatment resulted in cytotoxicity with an IC50 value of 13.27±0.66 μM in human A549 cells. Apoptosis was confirmed through Annexin V/PI staining and cell morphology observation. DH-22 induced apoptosis through the up-regulation of p53, Bax, cleaved caspase-9, and cleaved PARP, as well as the down-regulation of Bcl-2 expression (Figure 5). According to the findings of this investigation, DH-22 has a high potential for further development as a novel medicine.

Figure 5.
Figure 5.

Proposed mechanism of apoptosis-inducing renieramycin T derivative DH-22.

Acknowledgements

I.G.A is sincerely grateful to “Graduate Scholarship Programme for ASEAN and Non- ASEAN Countries Chulalongkorn University” for funding her Masters’ study in Thailand.

Footnotes

  • Authors’ Contributions

    Conceptualization, P.C.; validation, P.C., and Z.Z.E.; investigation, I.G.A., P.C., Z.Z.E., D.H., and M.Y.; resources, D.H., M.Y.; writing—original draft preparation, I.G.A.; writing—review and editing, P.C. All Authors have read and agreed to the published version of the manuscript.

  • Funding

    This research was supported by the National Research Council of Thailand (NRCT) (N41A640075).

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Received May 18, 2023.
  • Revision received June 16, 2023.
  • Accepted June 19, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Renieramycin T Derivative DH_22 Induces p53-dependent Apoptosis in Lung Cancer Cells
INDIANA GITA ANGGRAENI, ZIN ZIN EI, DAIKI HOTTA, MASASHI YOKOYA, PITHI CHANVORACHOTE
In Vivo Sep 2023, 37 (5) 1960-1966; DOI: 10.21873/invivo.13292
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