# Cepharanthine Promotes Ca2+-Independent Premature Red Blood Cell Death Through Metabolic Insufficiency and p38 MAPK/CK1α/COX/MLKL/PKC/iNOS Signaling

## TL;DR

Cepharanthine causes red blood cell death through a calcium-independent pathway involving several signaling molecules, which could impact its use in cancer treatment.

## Contribution

This study reveals a novel mechanism of CEP-induced eryptosis and hemolysis via Ca2+-independent signaling pathways.

## Key findings

- CEP increases phosphatidylserine exposure and hemolysis in red blood cells.
- CEP-induced eryptosis involves p38 MAPK, CK1α, COX, MLKL, PKC, and iNOS signaling.
- Modulating KCl efflux and intracellular calcium levels affects CEP's cytotoxic effects.

## Abstract

Nonspecific toxicity to normal and malignant cells restricts the clinical utility of many anticancer drugs. In particular, anemia in cancer patients develops due to drug-induced toxicity to red blood cells (RBCs). The anticancer alkaloid, cepharanthine (CEP), elicits distinct forms of cell death including apoptosis and autophagy, but its cytotoxicity to RBCs has not been investigated. Colorimetric and fluorometric techniques were used to assess eryptosis and hemolysis in control and CEP-treated RBCs. Cells were labeled with Fluo4/AM and annexin-V-FITC to measure Ca2+ and phosphatidylserine (PS) exposure, respectively. Forward scatter (FSC) was detected to estimate cell size, and extracellular hemoglobin along with lactate dehydrogenase and aspartate transaminase activities were assayed to quantify hemolysis. Physiological manipulation of the extracellular milieu and various signaling inhibitors were tested to dissect the underlying mechanisms of CEP-induced RBC death. CEP increased PS exposure and hemolysis indices and decreased FSC in a concentration-dependent manner with prominent membrane blebbing. Although no Ca2+ elevation was detected, chelation of intracellular Ca2+ by BAPTA-AM reduced hemolysis. Whereas SB203580, D4476, acetylsalicylic acid, necrosulfonamide, and melatonin inhibited both PS exposure and hemolysis, staurosporin, L-NAME, ascorbate, caffeine, adenine, and guanosine only prevented hemolysis. Interestingly, sucrose had a unique dual effect by exacerbating PS exposure and reversing hemolysis. Of note, blocking KCl efflux augmented PS exposure while aggravating hemolysis only under Ca2+-depleted conditions. CEP activates Ca2+-independent pathways to promote eryptosis and hemolysis. The complex cytotoxic profile of CEP can be mitigated by targeting the identified modulatory pathways to potentiate its anticancer efficacy.