Inhibition of ROS-NF-κB-dependent autophagy enhances Hypocrellin A united LED red light-induced apoptosis in squamous carcinoma A431 cells

https://doi.org/10.1016/j.cellsig.2020.109550Get rights and content

Abstract

Cutaneous squamous cell carcinoma (cSCC) is a type of malignant skin tumor derived from epidermal Malpighian cells. Photodynamic therapy is regarded as a crucial method in oncology. Hypocrellin A (HA), an efficient natural photosensitizer, has been reported to exert excellent light induced antiviral, antimicrobial and anticancer activity through mediating multiple signaling pathways. The purpose of the present study is to examine the effects of HA united red light irradiation on human squamous carcinoma A431 cells and further reveal the underlying regulatory mechanisms. The results showed that synergistic treatment of HA and red light irradiation inhibited cell proliferation and induced cell apoptosis and autophagy. Moreover, HA united red light irradiation caused a significant accumulation of reactive oxygen species (ROS), and induced the activation of c-Jun NH 2 terminal kinases (JNKs) which was inhibited by the antioxidant N-Acetyl-cysteine (NAC). Furthermore, HA united red light irradiation activated the nuclear factor-kappa B (NF-κB) pathway, and inhibition of NF-κB activity exacerbated HA united red light irradiation-induced apoptosis but suppressed cell autophagy. In addition, the inhibition of autophagy promoted HA united red light irradiation-induced apoptosis and facilitated the NF-κB activity. Over all, our results revealed that HA united red light irradiation could inhibit A431 cell proliferation by inducing apoptosis and autophagy via the activation of the ROS mediated JNK and NF-κB pathways, providing prospective for HA as a potential therapeutic for the treatment of cSCC.

Introduction

Cutaneous squamous cell carcinoma (cSCC), a type of malignant skin tumor derived from epidermal Malpighian cell, accounts for about 20% of non-melanoma skin tumors [1]. Prolonged exposure to ultraviolet (UV) and chemical carcinogens can increase the risk of cSCC [2]. In recent years, the incidence of cSCC has become more prevalent in the younger population [3]. Although cSCC is not life-threatening, if without treatment, it can grow large or transfer to other organs and induce aggressive cutaneous malignancy and serious complications. Over the past decades, a great deal of research has been carried out and considerable progresses have been achieved in the treatment of cSCC. Nevertheless, the cure rate of cSCC patients has not been significantly improved [4]. Recurrent cSCC is associated with an enhanced risk of aesthetic comorbidity, distant metastases and mortality. However, the traditional therapies, including surgical excision, radio-therapy, chemo-therapy and immune-therapy, may cause various damages to the treated skin area or the related organ functions [5]. Therefore, understanding the pathogenesis of cSCC and developing potential alternative therapies are necessary and urgent for the prevention and treatment of this disease.

Photodynamic therapy is regarded as a crucial method in oncology [6]. Visible light, photosensitizer and oxygen are indispensable to gain a therapy effect in photodynamic therapy. Red light has been widely applied in photodynamic therapy due to its capability to deeply puncture into the skin layer with little damage to it. Many clinical applications demonstrated that photosensitizer can be activated by red light. Red light united photosensitizer may achieve the photochemical effect via creating single oxygen and free radical, affecting the release of cytokines from macrophages or inhibiting angiogenesis. However, the precise mode of action is not yet fully known. Photosensitizers of pure natural products have obtained considerable attention from the field of medical application, on account of its unique biological activity, molecular structure diversity and hypobiotoxicity. Hypocrellin A (HA), isolated from S. bambusicola, is an efficient natural photosensitizer [7]. Many studies have shown that HA possesses prominent light-induced anticancer and antimicrobial activities, low dark toxicity and availability in a pure, monomeric form. Because the spectral absorption of HA nearly covers all of the light therapy window of superficial diseases, it is ideally suitable for the treatment of superficial disease [8]. When combined with visible light irradiation, the effect of hypocrellin was heightened due to the increased light energy. Studies have revealed that HA plays its cytotoxic roles through regulating a variety of signaling pathways [9,10]. For instance, photodynamic therapy with HA has been shown to exhibit antitumor activity via inhibiting cell viability and inducing tumor cell apoptosis [10]. Therefore, HA has been widely used as a photodynamic therapy agent against cancers [11,12].

The molecular basis of cSCC is complicated and covers both genetic and environmental elements (e.g., inflammatory responses, abnormal cytokine expression and UV exposure) [1]. To explore putative genes participated in the regulation of cSCC, we firstly studied the effects of HA combined with red light irradiation on apoptotic pathway, which is the classic mechanism of cell death induced by chemotherapy [13]. Furthermore, we also detected the impacts of HA combined with red light irradiation on the cell autophagy level. Autophagy is an evolutionarily conserved catabolic course and in most cases it occurs at constitutive levels in order to keep homeostasis [14]. Autophagy activation has been shown to be a crucial regulator for cancer development and progression, aging and neurodegenerative diseases [15]. However, the relationship between autophagy and apoptosis is intricate since autophagy can be involved in either cell survival or cell death, relying on the cell type and stimulus intensity. Understanding the mechanism underlying the interaction between autophagy and apoptosis in tumor cells is essential to identify new targets for tumor treatment and improve the efficiency of therapy. Reactive oxygen species (ROS) are mainly generated in mitochondria and play critical roles in mediating normal physiological processes and maintaining redox equilibrium [16]. Excessive production of ROS can give rise to oxidative injury to cells, especially malignant cells [17]. Increasing evidence supports that ROS are involved in mediation of mitogen-activated protein kinase (MAPK) signal transduction cascades, which are crucial players in the regulation of cell proliferation, apoptosis and autophagy [18]. In addition, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway is considered as another pivotal regulator involved in multiple aspects of proliferation and oncogenesis, including apoptosis, autophagy and inflammatory responses [19]. However, the detailed characterization among apoptosis, autophagy and NF-κB signaling pathways remains elusive.

In the present study, we designed to evaluate the cytotoxic effects of HA united red light irradiation on human squamous carcinoma A431 cells and reveal the underlying regulatory mechanisms. We found that HA united red light irradiation inhibited cell proliferation and induced cell apoptosis and autophagy. In addition, HA united red light irradiation caused a significant accumulation of reactive oxygen species (ROS), and induced the activation of c-Jun NH 2 terminal kinases (JNKs) which was inhibited by the antioxidant N-Acetyl-cysteine (NAC). Simultaneously, we evaluated the interplay between autophagy and NF-κB pathway in trigging cell apoptosis by means of inhibiting autophagy course and impeding NF-κB pathway, separately. Our results indicated that autophagy acts as a protective mechanism in A431 cells and that ROS might be the vital upstream signaling molecule of HA united red.

Section snippets

HA united red light irradiation inhibits cell proliferation in A431 cells

To examine whether HA united red light irradiation has a role in mediating cell proliferation, cell multiplication capacity was first measured by CCK-8 assay, a colorimetric assay for determining cell viability. The results showed that compared with the non-treated control group, A431 cells treated with HA united light irradiation at concentrations of 0–1 μM exhibited a remarkably decreased cell viability, while only a slight decrease in cell viability was detected in cells treated with HA or

Discussion

Cutaneous squamous cell carcinoma (cSCC), also called epidermal-like cancer, is a type of malignant skin tumor derived from epidermal Malpighian cells [1]. Over the past decades, a great deal of research has been carried out and considerable progresses have been achieved in the treatment of cSCC [4]. While most cSCC is cured with office-based therapy, including surgical excision, Mohs microscopic surgery, radio-therapy, electro-drying therapy, etc. The additional work-up and treatments

Conclusion

Collectively, our current studies show that HA united red light irradiation significantly inhibits cell growth, induces apoptosis and autophagy and activates NF-κB pathway in a ROS dependent pathway in A 431 cells (Fig. 8). There is a remarkable crosstalk between apoptosis, autophagy and NF-κB pathway. HA united red light induced autophagy may take a cytoprotective effect in maintaining the homeostasis of A431 cells, and inhibition of autophagy may effectively promote the therapeutic efficacy,

LED photosource

The output power of red light-emitting diode (LED)-red light source was tested by the Chinese National Institute of Metrology. The peak irradiation intensity at 630 nm is 300 μW/cm 2nm (Fig. S1). In our experiment, the power fluences delivered to a platform 20 mm under the LED red light arrays are 630 nm (5.68 mW/cm2). In our experiments, cells were irradiated at a distance of 20 mm to the LED array for 10 min.

Reagents and antibodies

Reagents 3- Methyladenine (3-MA), Acridine Orange (AO), N-acetyl cysteine (NAC), 2′,

Funding

This work was supported by the foundation of Military youth training program (grant no.16QNP024) and Beijing NOVA Program (grant no. Z161100004916151).

Author contributions

T.N. and Y.T. designed experiments, T.N. and G.G. performed all the experiments and analyzed the data, T.N. and Y.T. performed analyses and wrote the manuscript. Y.T. and Y.S. participated in guiding the article modification, and G.W. participated the experiments modified parts.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

References (41)

  • Z.J. He et al.

    Inhibiting ROS-NF-kappaB-dependent autophagy enhanced brazilin-induced apoptosis in head and neck squamous cell carcinoma

    Food Chem. Toxicol.

    (2017)
  • C. Zhai et al.

    Activation of AMPK prevents monocrotaline-induced pulmonary arterial hypertension by suppression of NF-kappaB-mediated autophagy activation

    Life Sci.

    (2018)
  • S. Qi et al.

    Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway

    Acta Pharm. Sin. B

    (2019)
  • R. Scherz-Shouval et al.

    Regulation of autophagy by ROS: physiology and pathology

    Trends Biochem. Sci.

    (2011)
  • Q. Zhang et al.

    30 years of NF-kappaB: a blossoming of relevance to human pathobiology

    CELL

    (2017)
  • R. Gnanajothy et al.

    A combined modality therapeutic approach to metastatic anal squamous cell carcinoma with systemic chemotherapy and local therapy to sites of disease: case report and review of literature

    J. Gastrointest. Oncol.

    (2016)
  • M. Yao et al.

    The research on lapatinib in autophagy, cell cycle arrest and epithelial to mesenchymal transition via Wnt/ErK/PI3K-AKT signaling pathway in human cutaneous squamous cell carcinoma

    J. Cancer

    (2017)
  • J. Quan et al.

    Potential molecular targets for inhibiting bone invasion by oral squamous cell carcinoma: a review of mechanisms

    Cancer Metastasis Rev.

    (2012)
  • K.A. Burton et al.

    Cutaneous squamous cell carcinoma: management of advanced and high-stage tumors

    Am. J. Clin. Dermatol.

    (2016)
  • L. Zhou et al.

    Multicolor imaging and the anticancer effect of a bifunctional silica nanosystem based on the complex of graphene quantum dots and hypocrellin A

    Chem. Commun. (Camb.)

    (2015)
  • Cited by (18)

    • Antifibrotic effects of Hypocrellin A combined with LED red light irradiation on keloid fibroblasts by counteracting the TGF-β/Smad/autophagy/apoptosis signalling pathway

      2021, Photodiagnosis and Photodynamic Therapy
      Citation Excerpt :

      Therefore, we attempted to clarify the impact of TGF-β1 on KFs from the perspective of autophagy. Our previous results demonstrated the potential role of HA-R-PDT in the suppression of proliferation in squamous carcinoma A431 cells; this treatment showed a synergistic effect compared to HA alone [22]. In the present study, we verified that HA-R-PDT suppressed cell proliferation and invasion and downregulated collagen synthesis and ECM accumulation.

    • EMT, cancer stem cells and autophagy; The three main axes of metastasis

      2021, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      According to the evidence, ROS is one of the factors involved in metastasis and invasion of cancer cells, and it exerts its effects by NF-κB–dependent autophagy. Studies performed in the recent years have revealed that inhibition of NF-κB–dependent autophagy makes the cancer cells sensitive to apoptosis [93,94]. On the other hand, induction of autophagy by inhibition of ROS–NF-κB signaling decreases the expression of MMP-2 and MMP-9, and it can be involved in inhibition of EMT [74,95].

    View all citing articles on Scopus
    View full text