Institute of Molecular Biosciences, University of Graz, Graz, Austria.
Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria.
J Cachexia Sarcopenia Muscle. 2024 Apr;15(2):562-574. doi: 10.1002/jcsm.13422. Epub 2024 Feb 1.
Cancer-associated cachexia (CAC) is a debilitating syndrome associated with poor quality of life and reduced life expectancy of cancer patients. CAC is characterized by unintended body weight reduction due to muscle and adipose tissue loss. A major hallmark of CAC is systemic inflammation. Several non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested for CAC treatment, yet no single medication has proven reliable. R-ketorolac (RK) is the R-enantiomer of a commonly used NSAID. The effect of RK on CAC has not yet been evaluated.
Ten- to 11-week-old mice were inoculated with C26 or CHX207 cancer cells or vehicle control (phosphate-buffered saline [PBS]). After cachexia onset, 2 mg/kg RK or PBS was administered daily by oral gavage. Body weight, food intake and tumour size were continuously measured. At study endpoints, blood was drawn, mice were sacrificed and tissues were excised. Immune cell abundance was analysed using a Cytek® Aurora spectral flow cytometer. Cyclooxygenase (COX) activity was determined in lung homogenates using a fluorometric kit. Muscle tissues were analysed for mRNA and protein expression by quantitative real-time PCR and western blotting analysis, respectively. Muscle fibre size was determined on histological slides after haematoxylin/eosin staining.
Ten-day survival rate of C26-bearing animals was 10% while RK treatment resulted in a 100% survival rate (P = 0.0009). Chemotherapy resulted in a 10% survival rate 14 days after treatment initiation, but all mice survived upon co-medication with RK and cyclophosphamide (P = 0.0001). Increased survival was associated with a protection from body weight loss in C26 (-0.61 ± 1.82 vs. -4.48 ± 2.0 g, P = 0.0004) and CHX207 (-0.49 ± 0.33 vs. -2.49 ± 0.93 g, P = 0.0003) tumour-bearing mice treated with RK, compared with untreated mice. RK ameliorated musculus quadriceps (-1.7 ± 7.1% vs. -27.8 ± 8.3%, P = 0.0007) and gonadal white adipose tissue (-18.8 ± 49% vs. -69 ± 15.6%, P = 0.094) loss in tumour-bearing mice, compared with untreated mice. Mechanistically, RK reduced circulating interleukin-6 (IL-6) concentrations from 334 ± 151 to 164 ± 123 pg/mL (P = 0.047) in C26 and from 93 ± 39 to 35 ± 6 pg/mL (P = 0.0053) in CHX207 tumour-bearing mice. Moreover, RK protected mice from cancer-induced T-lymphopenia (+1.8 ± 42% vs. -49.2 ± 12.1% in treated vs. untreated mice, respectively). RK was ineffective in ameliorating CAC in thymus-deficient nude mice, indicating that the beneficial effect of RK depends on T-cells.
RK improved T-lymphopenia and decreased systemic IL-6 concentrations, resulting in alleviation of cachexia and increased survival of cachexigenic tumour-bearing mice, even under chemotherapy and independent of COX inhibition. Considering its potential, we propose that the use of RK should be investigated in patients suffering from CAC.
癌症相关性恶病质（CAC）是一种衰弱综合征，与癌症患者的生活质量降低和预期寿命缩短有关。CAC 的特征是由于肌肉和脂肪组织丢失而导致非有意的体重减轻。CAC 的一个主要标志是全身炎症。已经有几种非甾体抗炎药（NSAIDs）被建议用于 CAC 治疗，但没有一种药物被证明是可靠的。R-酮咯酸（RK）是一种常用 NSAID 的 R-对映体。RK 对 CAC 的影响尚未得到评估。
10-11 周龄的小鼠接种 C26 或 CHX207 癌细胞或载体对照（磷酸盐缓冲盐水[PBS]）。恶病质发病后，每天通过口服灌胃给予 2mg/kg 的 RK 或 PBS。连续测量体重、食物摄入量和肿瘤大小。在研究终点时，抽取血液，处死小鼠并切除组织。使用 Cytek®Aurora 光谱流式细胞仪分析免疫细胞丰度。使用荧光试剂盒测定肺匀浆中环氧化酶（COX）活性。通过定量实时 PCR 和 Western blot 分析分别分析肌肉组织的 mRNA 和蛋白表达。在苏木精/伊红染色后对组织切片进行肌纤维大小测定。
C26 荷瘤动物的 10 天存活率为 10%，而 RK 治疗组的存活率为 100%（P=0.0009）。化疗后 14 天开始治疗的存活率为 10%，但在用 RK 和环磷酰胺联合治疗后所有小鼠均存活（P=0.0001）。存活率的提高与减轻 C26（-0.61±1.82 与-4.48±2.0 g，P=0.0004）和 CHX207（-0.49±0.33 与-2.49±0.93 g，P=0.0003）荷瘤小鼠体重减轻有关。与未治疗的小鼠相比，RK 改善了 C26（-1.7±7.1% 与-27.8±8.3%，P=0.0007）和 CHX207（-18.8±49% 与-69±15.6%，P=0.094）荷瘤小鼠的骼四头肌和性腺白色脂肪组织丢失。在机制上，RK 降低了 C26 荷瘤小鼠的循环白细胞介素-6（IL-6）浓度（从 334±151 至 164±123 pg/mL，P=0.047）和 CHX207 荷瘤小鼠的 IL-6 浓度（从 93±39 至 35±6 pg/mL，P=0.0053）。此外，RK 保护了小鼠免受癌症引起的 T 淋巴细胞减少症（与未治疗的小鼠相比，分别为+1.8±42%和-49.2±12.1%）。RK 在缺乏胸腺的裸鼠中对 CAC 无效，这表明 RK 的有益作用取决于 T 细胞。
RK 改善了 T 淋巴细胞减少症并降低了全身 IL-6 浓度，从而减轻了恶病质并增加了恶病质荷瘤小鼠的存活率，即使在化疗和 COX 抑制下也是如此。考虑到其潜力，我们建议在患有 CAC 的患者中研究 RK 的使用。
