The effects of Acyclovir administration to NCI-H1975 non-small cell lung cancer cells

doi:10.1016/j.tiv.2021.105301

Toxicology in Vitro

Volume 79, March 2022, 105301

https://doi.org/10.1016/j.tiv.2021.105301 Get rights and content

Highlights

•
ACV caused NCI-H1975 cell growth inhibition.
•
MMP reduction and altered mitochondrial size and shape were found.
•
Molecular analysis revealed significant mtDNA damage upon ACV treatment.
•
Mitochondria may be possible initial targets and/or sites of ACV cytotoxicity.

Abstract

The biochemical mechanisms by which the antiviral drug Acyclovir (ACV) may induce anticancer effects even without detecting human herpesviruses (HHVs) are still poorly understood. Herein, we investigated for the first time how NCI-H1975 non-small cell lung cancer cells responded in vitro to ACV administration by exploring mitochondrial damage and apoptosis induction. We confirmed ACV ability to cause the inhibition of cancer cell growth even without detecting intracellular HHVs; the drug also significantly inhibited the colony formation capacity of NCI-H1975 cells. Cell cycle analysis revealed an increase of the sub-G1 hypodiploid peak after ACV treatment; the activation of caspase-3 and the presence of DNA laddering sustained the capacity of the drug to induce apoptotic cell death. Regarding mitochondrial toxicity, a reduction of mitochondrial membrane potential, altered mitochondrial size and shape, and mtDNA damage were found after ACV administration. Furthermore, an increment of intracellular reactive oxygen species levels as well as the upregulation of NudT3 involved in DNA repair mechanisms were observed. Altogether, these findings suggest that mitochondria may be possible initial targets and/or sites of ACV cytotoxicity within cancer cells in the absence of intracellular HHVs.

Introduction

Acyclovir (ACV) is a synthetic guanosine analogue that has gained attention not only for its well documented antiviral activity against human herpesviruses (HHVs) (Schaeffer, 1982; Elion, 1993), but also for its potential application as an anticancer drug (Alibek et al., 2012). Indeed, other than its possible use in cytomegalovirus-positive glioblastoma (Solomon et al., 2014), other proposed mechanisms by which ACV could be employed in cancer therapy concern the inhibition of indoleamine 2,3-dioxygenase activity and of βTrCP1 ligase activity (Söderlund et al., 2010; Shafique and Rashid, 2017). The first enzyme is involved in developing immunosuppressive lymphocytes and immunologic tolerance in the tumour microenvironment (Godin-Ethier et al., 2011); the latter is involved in the targeted degradation of various growth and survival factors via ubiquitin-mediated pathway (Frescas and Pagano, 2008). Accordingly, the in vitro suppressive effect of ACV on MCF-7 breast cancer cells, Jurkat leukaemia cells, and U87 MG glioblastoma cells has been recently reported (Shaimerdenova et al., 2017; Benedetti et al., 2018; Özdemir and Göktürk, 2019), demonstrating ACV ability to affect cancer cell viability and sustaining its potential as adjuvant therapy in cancer treatment.

The relevant biochemical mechanisms by which ACV may induce anticancer effects even without the detection of HHVs (Benedetti et al., 2018; Özdemir and Göktürk, 2019) that could mediate the intracellular phosphorylation of ACV via viral kinases rendering it capable of blocking DNA synthesis (Schaeffer, 1982; Elion, 1993), are still poorly understood. Early studies showed that low detectable phosphorylated ACV levels could be present within cells even in the absence of viral thymidine kinases (McMahon et al., 2011), indicating that endogenous kinases could phosphorylate the drug. In fact, as a nucleoside analog, ACV could be involved in the nucleoside salvage pathway and might be activated by intracellular nucleoside kinases and inhibit DNA synthesis (Jordheim et al., 2013). Moreover, even if the high fidelity of human replicative DNA polymerases tends to limit the nuclear DNA incorporation of chain-terminating nucleoside analogs such as ACV, whereas the viral polymerases allow their incorporation into viral genomes, due to the large size of the human genome, its exclusion is not absolute (Nickel et al., 1992). At the same time, antiviral-induced mitochondrial toxicity has been reported (Huang et al., 2013) and attributed to the less stringent selectivity of mitochondrial DNA polymerase against antiviral nucleoside analogs (Johnson et al., 2001).

Following our previous research experience on ACV administration to leukemic cells (Benedetti et al., 2018), in the present study, we proposed to further investigate in vitro how cancer cells responded to ACV treatment by exploring for the first time the effects of antiviral drug administration to NCI-H1975 non-small cell lung cancer cells, the most common subtype of lung cancer (85% of all lung cancers). Particular focus was given to understanding the biochemical mechanisms underlying ACV-induced mitochondrial damage and apoptotic cell death.

Section snippets

Cell culture conditions and drug treatment

NCI-H1975 cells (ICLC, Genova, Italy) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, and 1% penicillin/streptomycin 100 U/ml, and maintained in a CO₂ incubator at 37 °C and 5% CO₂. Cell culture materials were from VWR International (Milan, Italy), while reagents from Sigma-Aldrich (Milan, Italy).

ACV (Recordati, Milan, Italy) was resuspended in 0.9% NaCl and sterilized using a 0.45 μm syringe-filter before use. Clinically relevant

Results and discussion

The in vitro suppressive effect of ACV on MCF-7 breast cancer cells, Jurkat leukaemia cells, and U87 MG glioblastoma cells has been recently reported (Shaimerdenova et al., 2017; Benedetti et al., 2018; Özdemir and Göktürk, 2019); however, the biochemical mechanisms underlying ACV anticancer effects are still poorly understood. Further investigating how cancer cells responded to the antiviral drug, in the present study, we observed for the first time a dose-dependent inhibition of NCI-H1975

Conclusions

In the present study, we observed the inhibition of cell proliferation by ACV administration to NCI-H1975 lung cancer cells without detecting intracellular HHVs. The activation of caspase-3 and the presence of nuclear DNA laddering in ACV-treated cells sustain drug's ability to cause apoptotic cell death. The reduction of mitochondrial membrane potential, the alterations of mitochondrial morphology, and the occurrence of mtDNA damage suggest mitochondria as possible initial targets and/or sites

Declaration of Competing Interest

Francesco Palma reports financial support was provided by non-profit organization R.U.O.T.A. (Rapallo, GE, Italy).

Acknowledgments

We are grateful to the non-profit organization R.U.O.T.A. (Rapallo, GE, Italy) which funded the research.

References (43)

S. Benedetti et al.
Acyclovir induces cell cycle perturbation and apoptosis in Jurkat leukemia cells, and enhances chemotherapeutic drug cytotoxicity
Life Sci.
(2018)
K.R. Beutner
Valacyclovir: a review of its antiviral activity, pharmacokinetic properties, and clinical efficacy
Antivir. Res.
(1995)
M.M. Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)
S. Catalani et al.
Reduced cell viability and apoptosis induction in human thyroid carcinoma and mesothelioma cells exposed to Cidofovir
Toxicol. in Vitro
(2017)
S. Dasari et al.
Cisplatin in cancer therapy: molecular mechanisms of action
Eur. J. Pharmacol.
(2014)
F. Farabegoli et al.
Betalains increase vitexin-2-O-xyloside cytotoxicity in CaCo-2 cancer cells
Food Chem.
(2017)
C. Ginestier et al.
ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome
Cell Stem Cell
(2007)
C. Guidi et al.
Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA
Biochim. Biophys. Acta
(2008)
H. Izzedine et al.
Antiviral drug-induced nephrotoxicity
Am. J. Kidney Dis.
(2005)
A.A. Johnson et al.
Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase
J. Biol. Chem.
(2001)

W. Nickel et al.

Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase

J. Biol. Chem.

(1992)

H.J. Schaeffer

Acyclovir chemistry and spectrum of activity

Am. J. Med.

(1982)

S. Shafique et al.

Antiviral drug acyclovir exhibits antitumor activity via targeting βTrCP1: molecular docking and dynamics simulation study

J. Mol. Graph. Model.

(2017)

Y. Songyang et al.

Effect of vitamin D on malignant behavior of non-small cell lung cancer cells

Gene

(2021)

K. Alibek et al.

Using antimicrobial adjuvant therapy in cancer treatment: a review

Infect Agent Cancer.

(2012)

J. Carreras-Puigvert

A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family

Nat. Commun.

(2017)

S. Catalani et al.

Metabolism modifications and apoptosis induction after CellfoodTM administration to leukemia cell lines

J. Exp. Clin. Cancer Res.

(2013)

S. Catalani et al.

Oxidative stress and apoptosis induction in human thyroid carcinoma cells exposed to the essential oil from Pistacia lentiscus aerial parts

PLoS One

(2017)

S.K. Chiou et al.

Survivin - an anti-apoptosis protein: its biological roles and implications for cancer and beyond

Med. Sci. Monit.

(2003)

G.B. Elion

Acyclovir: discovery, mechanism of action, and selectivity

J. Med. Virol. Suppl.

(1993)

N.A. Franken et al.

Clonogenic assay of cells in vitro

Nat. Protoc.

(2006)

Cited by (5)

New insights into the cytotoxic effects of Thymus vulgaris essential oil on the human triple-negative breast cancer cell line MDA-MB-231
2023, Toxicology in Vitro
Essential oils (EOs) are natural products that have gained wide interest due to their biological activities and anticancer properties through various mechanisms. The present study aimed to test the cytotoxicity of Thymus vulgaris L. (thyme) EO of Italian origin, rich in thymol (49.6%) and p-cymene (18.8%), towards the triple-negative breast cancer cell line MDA-MB-231 and to investigate the biochemical mechanisms underlying its antitumor activity. Thyme EO reduced cancer cell viability in a dose-dependent manner after 24 h treatment, with an IC₅₀ value equal to 75.1 ± 15.2 μg/ml; simultaneously, the inhibition of cancer cell migration and colony formation capacity was evidenced. Thyme EO antiproliferative effects were related to the induction of apoptosis as demonstrated by the increased expression of the pro-apoptotic proteins Bax, cleaved caspase-3, phospho-p53, and SMAC/Diablo and by the reduction of the anti-apoptotic proteins Bcl-2, cIAP-1, cIAP-2, HIF-1α, survivin, and XIAP. Thyme EO administration led to the early formation of intracellular ROS, followed by the increment of MDA as an index of lipid peroxidation and by the decreased expression of the antioxidant enzymes catalase and PON2. The upregulation of Nrf2 mRNA expression and the strong induction of HO-1 sustained the activation of the Nrf2 pathway by thyme EO. These data showed that the EO from Thymus vulgaris L. might inhibit the malignant phenotype of MDA-MB-231, thus suggesting potential benefits against human triple-negative breast cancer.
Mitochondrial DNA-targeted therapy: A novel approach to combat cancer
2023, Cell Insight
Mitochondrial DNA (mtDNA) encodes proteins and RNAs that are essential for mitochondrial function and cellular homeostasis, and participates in important processes of cellular bioenergetics and metabolism. Alterations in mtDNA are associated with various diseases, especially cancers, and are considered as biomarkers for some types of tumors. Moreover, mtDNA alterations have been found to affect the proliferation, progression and metastasis of cancer cells, as well as their interactions with the immune system and the tumor microenvironment (TME). The important role of mtDNA in cancer development makes it a significant target for cancer treatment. In recent years, many novel therapeutic methods targeting mtDNA have emerged. In this study, we first discussed how cancerogenesis is triggered by mtDNA mutations, including alterations in gene copy number, aberrant gene expression and epigenetic modifications. Then, we described in detail the mechanisms underlying the interactions between mtDNA and the extramitochondrial environment, which are crucial for understanding the efficacy and safety of mtDNA-targeted therapy. Next, we provided a comprehensive overview of the recent progress in cancer therapy strategies that target mtDNA. We classified them into two categories based on their mechanisms of action: indirect and direct targeting strategies. Indirect targeting strategies aimed to induce mtDNA damage and dysfunction by modulating pathways that are involved in mtDNA stability and integrity, while direct targeting strategies utilized molecules that can selectively bind to or cleave mtDNA to achieve the therapeutic efficacy. This study highlights the importance of mtDNA-targeted therapy in cancer treatment, and will provide insights for future research and development of targeted drugs and therapeutic strategies.
Targeting the mitochondria in chronic respiratory diseases
2022, Mitochondrion
Citation Excerpt :
Dolutegravir (DTG) is a first line drug for Acquired Immune Deficiency Syndrome (AIDs) management, which can increase the ROS levels and intracellular calcium levels. DTHP, or known as 7-methoxy-4-methyl-6,8-dioxo-N-(3-(1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)phenyl) 3, 4, 6, 8, 12, 12a-hexahydro-2H-pyrido [1′,2′:4,5] pyrazino (Schumacker et al., 2014; Aravamudan et al., 2013; Schumacker et al., 2014; Cloonan and Choi, 2016; Piantadosi and Suliman, 2017; Crichton et al., 2017; Harris et al., 2012; Kessler et al., 1976; Balaban, 2009; Nicholls, 1978; Klingenberg, 2008; Halestrap and Richardson, 2015; Fang et al., 2019; Chellappan et al., 2021; Mehta et al., 2020; Diseases and Injuries, 2020; Manandhar et al., 2022; Mehta et al., 2021; Chan et al., 2021; Alamil et al., 2022; Kim et al., 2020; Dhanjal et al., 2022; Allam et al., 2022; Zifa et al., 2012; Dada and Sznajder, 2011; Rowlands et al., 2011; Sathish et al., 2011; Aravamudan et al., 2014; Simoes et al., 2012; Phaniendra et al., 2015; Delmotte and Sieck, 2015; Ilmarinen-Salo et al., 2012; Mabalirajan et al., 2013; Shastri et al., 2021; Konga et al., 2009; Moir et al., 2011; Xu et al., 2010; Tian et al., 2019; Ghavami et al., 2010; Wei et al., 2019; Wang et al., 2019; Silveira et al., 2019; Mabalirajan et al., (1985). 2009,; Karaman et al., 2021; Mabalirajan et al., 2009; Mabalirajan et al., 2010; Kumar et al., 2013; Yao et al., 2018; Michaeloudes et al., 2021; Grafton et al., 2014; Tan et al., 2022; Chen et al., 2021; Khursheed et al., 2022; Bhavna et al., 2009; Kong et al., 2008; Chen et al., 2012; Inapagolla et al., 2010; Chan et al., 2021; Paudel et al., 2022; Mehta et al., 2021; Paudel et al., 2021; Mehta et al., 2021; Wadhwa et al., 2021; Solanki et al., 2020; Qin et al., 2021; Paudel and Kim, 2020; Mabalirajan et al., 2013; Cui et al., 2019;112:108694.; Gheware et al., 2021; Barnes, 2019; Barnes, 2017; Lerner et al., 2016; Paudel et al., 2021; Paudel et al., 2022; Liu et al., 2022; Wiegman et al., 2015; Hoffmann et al., 2019; Ryter et al., 2012; Ornatowski et al., 2020; Nakahira et al., 2014; Jiang et al., 2019; Araya et al., 2019; Yue and Yao, 2016; Ito et al., 2015; Ng Kee Kwong et al., 2017; Tsubouchi et al., 2018; Hikichi et al., 2019; Ryter and Choi, 2015; Mizumura et al., 2014; Sharma et al., 2021; Hoffmann et al., 2013; Michaeloudes et al., 2017;14(Supplement_5):S374–S82.; Aravamudan et al., 2017; Jiang et al., 2017; Hawkins and Mora, 2017; Mariani, 2016; Sagar et al., 2021; Michaeloudes et al., 2011; Zuo and Wijegunawardana, 2021; Bialas et al., 2016; Puente-Maestu et al., 2009; Belchamber et al., 2019; Birch et al., 2018; Paudel et al., 2022; Summer et al., 2019; Puente-Maestu et al., 2009; Naimi et al., 2011; Perez-Rial et al., 2020; Marin-Corral et al., 2009; Pouwels et al., 2016; Zhang et al., 2018; Pouwels et al., 2014; Larson-Casey et al., 2020; Nucera et al., 2022; Devkota et al., 2021; Barnes, 2020; Mahalanobish et al., 2020; Baker et al., 2020; Even et al., 2018; Dong and Zhu, 2014; Li et al., 2013; Beijers et al., 2018; Wecht and Rojas, 2016; Gu et al., 2015; Shigemura et al., 2006; Guan et al., 2013; Antunes et al., 2014; Peron et al., 2015; Song et al., 2014; Hiemstra, 2013; Dong et al., 2015; Mehta et al., 2021; Li et al., 2018; Li et al., 2014; Maremanda et al., 2019; Sung et al., 2020; Siegel et al., 2022; Malyla et al., 2020; Siegel et al., 2018; Paudel et al., 2020; Dasgupta et al., 2012; Warburg, 1956; Plas and Thompson, 2005; Chiche et al., 2010; de Moura et al., 2010; Servais et al., 2003; Zhang et al., 2021; Aman et al., 2020; Hardwick et al., 2021; Liu et al., 2019; Yuan et al., 2020; Patel et al., 2022; Tian et al., 2018; Carra et al., 2021; Bai and Jiao, 2020; Allaway et al., 2018; Lee et al., 2019; Deribe et al., 2018; Martin et al., 2017; Mehta et al., 2021; D'Almeida et al., 2019; Cho and Kleeberger, 2020; Kazdal et al., 2017; Li et al., 2019; Moreno et al., 2020; Murray et al., 2018; Kim et al., 2021; Henson et al., 2017; Magraner-Pardo et al., 2021; Prakasam et al., 2017; Ye et al., 2021; Xie et al., 2020; Wang et al., 2020; Erkisa et al., 2021; Wang et al., 2020; Park et al., 2021; Yin et al., 2021; Rajendran et al., 2021; Tong et al., 2021; Chang et al., 2020; Elborn, 2016; Chellappan et al., 2020; Patergnani et al., 2020; Feigal and Shapiro, 1979; Shapiro et al., 1979; Valdivieso et al., 2007; Rimessi et al., 2015; Atlante et al., 2016; Manna et al., 2000; Xu et al., 2012; Hamdaoui et al., 2011; Dhooghe et al., 2015; Cho et al., 2019; Smith et al., 2003; Zang et al., 2012; Sokol et al., 1989; Matsuda et al., 2013; Manevski et al., 2020; Sun et al., 2019; Bernard et al., 2017; Jiang et al., 2017; Maremanda et al., 2021; Sundar et al., 2019; Zhang et al., 2019; Zhang et al., 2020; Swartzendruber et al., 2020; Nam et al., 2017; Zhang et al., 2018; Wang et al., 2019; Marullo et al., 2013; Kursunluoglu et al., 2018; Takenaka et al., 2017; Puri et al., 2020; Vitiello et al., 2018; Li et al., 2005; Cheng et al., 2019; Huang et al., 2018; Shieh et al., 2017; Yilmaz et al., 2018; Benedetti et al., 2022; Zhang et al., 2019; Zhao et al., 2020; Liu et al., 2018; Wang et al., 2021; Chen et al., 2022; Zhao et al., 2018; Chai et al., 2020; Xue et al., 2020; Aravamudan et al., 2013; Cloonan and Choi, 2016) oxazine-9-carboxamide is a derivative of DTG, and its anti-tumour properties were studied. DTHP is non-cytotoxic to healthy cells, it can inhibit the colony-forming ability and the proliferation of NSCLC cells.
Mitochondria are one of the basic essential components for eukaryotic life survival. It is also the source of respiratory ATP. Recently published studies have demonstrated that mitochondria may have more roles to play aside from energy production. There is an increasing body of evidence which suggest that mitochondrial activities involved in normal and pathological states contribute to significant impact to the lung airway morphology and epithelial function in respiratory diseases such as asthma, COPD, and lung cancer. This review summarizes the pathophysiological pathways involved in asthma, COPD, lung cancer and highlights potential treatment strategies that target the malfunctioning mitochondria in such ailments. Mitochondria are responsive to environmental stimuli such as infection, tobacco smoke, and inflammation, which are essential in the pathogenesis of respiratory diseases. They may affect mitochondrial shape, protein production and ultimately cause dysfunction. The impairment of mitochondrial function has downstream impact on the cytosolic components, calcium control, response towards oxidative stress, regulation of genes and proteins and metabolic activities. Several novel compounds and alternative medicines that target mitochondria in asthma and chronic lung diseases have been discussed here. Moreover, mitochondrial enzymes or proteins that may serve as excellent therapeutic targets in COPD are also covered. The role of mitochondria in respiratory diseases is gaining much attention and mitochondria-based treatment strategies and personalized medicine targeting the mitochondria may materialize in the near future. Nevertheless, more in-depth studies are urgently needed to validate the advantages and efficacy of drugs that affect mitochondria in pathological states.
A microfluidic approach to fabricate sucrose decorated liposomes with increased uptake in breast cancer cells
2022, European Journal of Pharmaceutics and Biopharmaceutics
Citation Excerpt :
Eventually, cells were rinsed with acetic acid 1 % as many times to remove the unincorporated color. To solubilize the incorporated dye, 10 mM tris was added and the absorbance was measured at 570 nm in a microplate reader (Multiskan FC, Thermo Scientific) [42]. Data were expressed as a percentage (%) versus non-treated cells (controls).
Developing targeted drug delivery systems is an urgent need to decrease the side effects and increase the drug's efficiency. Most cancer cells show an increased sugar consumption compared to healthy cells due to the deregulation of sugar transporters. Consequently, liposomes, as a biocompatible nanocarrier, could be surface decorated by sugars to enhance drug targeting into cancer cells. Our work outlines a new strategy to easily manufacture sucrose decorated liposomes using sucrose stearate, a biocompatible and biodegradable non-ionic surfactant, with a scalable microfluidic approach. Sucrose decorated liposomes were loaded with berberine hydrochloride, a well-known phytochemical compound to investigate its effects on triple-negative breast cancer cells (MDA-MB-231). Using the microfluidic manufacturing system, we prepared berberine-loaded liposomes using a mixture of phosphatidylcholine and cholesterol with and without sucrose stearate with a size up to 140 nm and narrow polydispersity. Stability was confirmed for 90 days, and the in vitro release profile was evaluated. The formulations showed acceptable in vitro biocompatibility and significantly higher anti-proliferative effect on MDA-MB-231 cell line. These results have been confirmed by an increased uptake evaluated by flow cytometry and confocal microscopy. Taken together, our findings represent an innovative, easy, and scalable approach to obtain sugar decorated liposomal formulations without any surface-chemistry reactions. They can be potentially used as an anticancer targeted drug delivery system.
Successful examples of blockbusters drugs for drug repurposing
2023, Drug Repurposing

View full text

The effects of Acyclovir administration to NCI-H1975 non-small cell lung cancer cells

Highlights

Abstract

Introduction

Section snippets

Cell culture conditions and drug treatment

Results and discussion

Conclusions

Declaration of Competing Interest

Acknowledgments

Life Sci.

Antivir. Res.

Anal. Biochem.

Toxicol. in Vitro

Eur. J. Pharmacol.

Food Chem.

Cell Stem Cell

Biochim. Biophys. Acta

Am. J. Kidney Dis.

J. Biol. Chem.

J. Biol. Chem.

Am. J. Med.

J. Mol. Graph. Model.

Gene

Using antimicrobial adjuvant therapy in cancer treatment: a review

Infect Agent Cancer.

A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family

Nat. Commun.

Metabolism modifications and apoptosis induction after CellfoodTM administration to leukemia cell lines

J. Exp. Clin. Cancer Res.

Oxidative stress and apoptosis induction in human thyroid carcinoma cells exposed to the essential oil from Pistacia lentiscus aerial parts

PLoS One

Survivin - an anti-apoptosis protein: its biological roles and implications for cancer and beyond

Med. Sci. Monit.

Acyclovir: discovery, mechanism of action, and selectivity

J. Med. Virol. Suppl.

Clonogenic assay of cells in vitro

Nat. Protoc.