Cathepsin B: Plasma Expression and Concentration in Non-Hodgkin Lymphoma Patients

Abstract

Numerous studies point to the significance of cathepsin B (CTSB) in the development of carcinoma. Therefore, the aim of this pilot study was to investigate the levels of cathepsin B (CTSB) and the expression of CTSB mRNA in the plasma of non-Hodgkin lymphoma (NHL) patients. Methods: The study included 44 newly diagnosed NHL patients and 35 healthy volunteers comprising the control group. CTSB in the plasma samples were detected using the enzyme-linked immunosorbent assay (ELISA). Results: The level of CTSB was significantly higher in NHL patients compared to control subjects: 15.28 (11.68–17.23) versus 11.57 (10.12–13.41), p = 0.003. In addition, a positive correlation between plasma CTSB mRNA and CTSB after therapy was observed (rho = 0.591, p = 0.026). Regarding redox parameters, we found a negative correlation between CTSB and the total antioxidant status (TAS) (rho = −0.499, p = 0.035), as well as a positive correlation with the total oxidant status (TOS) (rho = 0.576, p = 0.012). Conclusions: Targeting CTSB might have significant clinical relevance in the diagnostics of NHL.

1. Introduction

Non-Hodgkin lymphomas (NHLs) are a diverse category of lymphoproliferative cancers that are more likely to spread to extranodal sites and are significantly less predictable than Hodgkin’s lymphomas [1]. Ninety percent of NHL malignancies are mature B-cell non-Hodgkin lymphomas (B-cell NHLs), and their incidence has been steadily rising over time. Follicular lymphoma (approximately 20%) and diffuse large B-cell lymphoma (about 30%) are by far the most prevalent NHL subtypes [2]. Plasma cell myeloma (multiple myeloma), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and marginal zone lymphoma (MZL) are other types of B-cell lymphoma, though their incidence and frequency vary. Plasma cell myeloma is a relatively rare type, accounting for only around 1% of all cancers and 10% of all hematologic malignancies. Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) accounts for roughly 7% of newly diagnosed non-Hodgkin’s lymphoma (NHL), while marginal zone lymphoma accounts for about 5–10% of all non-Hodgkin lymphoma cases [3].

The etiology of NHL remains uncertain despite the large number of factors studied. Several research have demonstrated that dysregulation of redox homeostasis contributes to the development of B-cell cancers [4]. Despite that, among the factors that might be involved in the development of lymphoproliferative disorders are members of the cysteine proteases family [5].

Among proteases, a large group of enzymes that catalyze the cleavage of peptide linkages in proteins, cathepsin B (CTSB) draws significant interest from researchers. CTSB (EC 3.4.22.1) is the first and currently the best-characterized member of the C1 family of papain-like, lysosomal cysteine peptidases [6]. Several lines of studies confirmed that in patients with B-cell lymphoma, the main source of CTSB in blood is likely to be tumor cells (malignant B cells) and immune cells associated with the tumor microenvironment, such as macrophages and monocytes [5,7]. Under normal physiological conditions, CTSB is primarily found in the acidic lysosomal compartment where it is essential for controlling various cellular functions such as tissue remodeling, protein degradation, regulation of pro-hormone and pro-enzyme activation, antigen processing, inflammatory responses and apoptosis [5]. On the other hand, the damaging of lysosome integrity and rupturing them by oxidative stress can affect the cathepsin subcellular location [8]. It may release the proteins into the cytosol where they can promote apoptosis, tumor invasion and metastasis (Figure 1).

The CTSB pre-pro-enzyme will translocate into the lumen of the rough endoplasmic reticulum, where the inactive precursor, pro-cathepsin B (shown by a purple symbol), is synthesized. Pro-CTSB is subsequently translocated via the rough endoplasmic reticulum to the Golgi apparatus, where it undergoes autocatalytic activation, resulting in the conversion to mature (active) CTSB (red symbol) through proteolytic cleavage and pro-peptide dissociation. Secreted active proteolytic CTSB may participate in the remodeling and degradation of extracellular matrix components, triggering caspase cascades that induce both cell apoptosis and extracellular matrix breakdown. Conversely, the truncated CTSB (blue symbol) is not translocated to the endolysosomal compartment but is instead localized to the mitochondria. The potential interaction between truncated CTSB and regulatory proteins on mitochondria may induce nuclear fragmentation and subsequent cellular death through its non-proteolytic functions. CTSB, cathepsin B; RER, rough endoplasmic reticulum; ECM, extracellular matrix.

The review published recently emphasized the critical role that reactive oxygen species (ROS) play in controlling the activity of a particular class of proteases called cathepsins [9]. Accumulated data reported that oxidative stresses are associated with CTSB activity [8].

There is growing evidence suggesting that both CTSB and impaired redox homeostasis are involved in apoptosis at multiple levels, which is responsible for the onset and progression of numerous diseases [6,10]. Moreover, the impaired redox homeostasis and CTSB in the tumor microenvironment can affect prognosis and response to therapy [11,12]. In addition, CTSB expression is upregulated by oxidative stress [8,13].

Multiple lines of evidence suggest that CTSB is upregulated across a variety of cancers and has been shown to be closely related to the development and metastasis of cancer [14,15]. Increases in expression at the mRNA and protein levels, increased activity and altered trafficking (localization and secretion) of CTSB have been found to correlate with different malignancies [16]. Expression and activity of CTSB have been correlated with a number of pathologies, including cancer, arthritis, cardiovascular disease and Alzheimer’s disease [17,18,19,20]. Therefore, CTSB has become increasingly recognized as a promising target in tumor imaging and cancer treatment.

The final level of regulation of CTSB activity is by endogenous inhibitors such as cystatins [21]. Cystatin C, also known as Cys C, is an inhibitor of cysteine proteinase that is essential for controlling the breakdown of intracellular and extracellular proteins. According to earlier studies, Cys C has a role in changing the proteolytic system in cancer, which might explain why patients with increased plasma Cys C levels indicate a worse prognosis [17,18]. Considering that there are currently no good biochemical markers for early diagnosis and for monitoring the treatment success of patients with non-Hodgkin’s lymphoma, our goal was to analyze whether cathepsin B could have the potential to be a marker. We also analyzed the association of this protein with the expression of its gene as well as with current markers of oxidative stress.

2. Material and Methods

2.1. Subjects

This pilot study comprised 79 participants and was carried out at the University Clinical Centre of the Republic of Srpska, Banja Luka (Bosnia and Herzegovina). A total of 44 newly diagnosed patients with non-Hodgkin’s B lymphoma and 35 healthy volunteers comprising the control group were admitted to the University Clinical Centre of the Republic of Srpska during the same period of time for either routine checkup. The study’s inclusion criteria, which were fulfilled by all patients who were over the age of 18, were that they had no prior history of cancer and had lymphoma as their primary malignancy. The control group comprised 35 matched healthy volunteers who satisfied the following inclusion criteria: both genders, no history of malignancy, chronic disease, or acute infection in the previous 3 months, clinical laboratory parameters within the reference range and non-pregnancy or lactation status. NHL patients were treated in accordance with international lymphoma guidelines, which were adopted at the national level due to drug availability. All NHL patients received first-line immuno-chemotherapies, such as the R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) or an equivalent regimen, such as R-EPOCH (rituximab, etoposide, doxorubicin, vincristine, prednisone cyclophosphamide) Immunochemotherapy began, following a baseline FDG-PET/CT lymphoma staging.

2.2. Laboratory Analysis

Blood samples were taken from patients after an overnight fast (first time-point) and after immunochemotherapy treatment (reassessment point, second time-point). Samples were centrifuged promptly upon venipuncture, and the plasma and serum were isolated and stored at −80 °C until analysis.

CTSB levels were determined using the ELISA (Enzyme-Linked Immunosorbent Assay) (ab119584 Cathepsin B Human ELISA Kit; Abcam plc. Cambridge, GB, UK) according to the manufacturer’s instructions. In short, plasma, diluted 1:10, was added to microtiter plate wells pre-coated with the monoclonal antibody for CTSB detection. The lowest detectable dosage of human CTSB was 5 ng/mL. The standard curve and triplicate samples showed a coefficient of variation (CV) of less than 10% for intra-assay and less than 15% for inter-assay.

Cystatin C was measured using the Cobas e 801 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The total antioxidant status (TAS) and total oxidant status (TOS) were determined using an ILab 300+ (Instrumentation Laboratory, Milan, Italy) [22].

Only 18 patients were followed up until the end of the therapy. In this group, CTSB mRNA expression was performed. The total ribonucleic acid (RNA) was isolated from plasma by the commercial kit, miRNeasy^® serum/plasma Kit (Qiagen, Hilden, Germany). Reverse transcription was performed according to manufacturer recommendations using the High-capacity complementary deoxyribonucleic acid reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantification of CTSB gene expression levels were performed using TaqMan^® 5′-nuclease gene expression assays (Applied Biosystems, Foster City, CA, USA) for the human cathepsin B gene (Hs00947439_m1). Cathepsin B gene expression levels were expressed as relative levels and were normalized using GAPDH as a reference gene using following equations:

Relative gene expression levels for cathepsin B = 2^−ΔCt = 2^{−(Ct Cathepsin B − Ct GAPDH)}

In equations, Ct represented the cycle threshold that was obtained for each sample.

Negative controls for reverse transcription (sample without reverse transcriptase and sample without RNA) and non-template control (sample without complementary DNA) were included in each plate during experiments. Gene expression analysis was performed using 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

2.3. Statistical Analysis

The data distribution was tested using the Kolmogorov–Smirnov test. Depending on the normality of the distribution, the Mann–Whitney U test was used to analyze the differences in the data between patients and controls. The correlations between the variables were estimated using Spearman’s correlation coefficient (rho). A sensitivity analysis was performed using a rank-based regression model to assess the robustness of the observed correlations. The median for independent data and the median of difference for dependent data with an interquartile range were presented for non-normally distributed variables. A power analysis was performed to justify the sample size. The statistical analyses were performed with PASW Statistics, v. 27, software (Chicago, IL, USA) and R software. A two-tailed p-value ≤ 0.05 was considered statistically significant.

3. Results

3.1. Demographic and Clinical Data of NHL Patients and Controls

The median age of control subjects, with an interquartile range of 49 (36–55), was substantially younger than that of NHL patients, whose median age was 65 (55–72), p < 0.001. The correlation analysis determined that there is no statistically significant relationship between age and CTSB (r = 0.192; p = 0.061). The gender distribution varied widely between groups; males were more prevalent in NHL patient groups compared to the control group (p = 0.025). The most common NHL subtype was follicular lymphoma (47.73%), followed by diffuse large B-cell lymphoma (DLBCL) (31.81%) and small lymphocytic lymphoma (SLL) (9.10%).

3.2. Plasma Cathepsin B, Cystatin C and Redox Parameters in NHL Patients and Control Groups

The value of CTSB was detected in the plasma of NHL patients and the healthy population. In addition, Cys C, TOS and TAS levels of patients with NHL and healthy controls were determined. Results are listed in

Table 1. Comparison of biomarkers of healthy subjects and NHL patients.

	Controls, n = 35	NHL Patients, n = 44	p
CTSB (ng/mL)	11.57 (10.12–13.41)	15.28 (11.68–17.23)	0.003
Cys C (mg/L)	0.82 (0.74–0.90)	1.03 (0.88–1.24)	<0.001
TAS (mmol/L)	1147 (1002–1222)	998 (818–1144)	0.014
TOS (mmol/L)	9.30 (5.6–12.80)	12.60 (8.2–17.40)	0.025

Compared by Mann—Whitney U test. Abbreviations: CTSB, cathepsin B; Cys C, cystatin C; TAS, total oxidant status; TOS, total antioxidant status; p, level of significance.

. Plasma CTSB levels of the patients were significantly higher (p = 0.003) than those of the control group. Cys C levels of the patient group were also significantly higher (p < 0.001) compared to the controls. In addition, the assessment of the redox parameters showed that the TAS values were significantly reduced, in contrast to the TOS parameter which had significantly higher concentrations in the NHL group, as compared to the control group.

3.3. The Association Between CTSB Plasma Concentration and NHL Subtype

In the following part of the study, we categorized the patients into high- and low-grade NHL. The high-grade NHL subtype comprised 14 DLBCL and 2 mantle cell lymphoma patients, while the low-grade NHL subtype consisted of 21 FL and 5 small lymphocytic lymphoma patients. There was no significant difference in CTSB concentration between the high-grade and low-grade NHL subtype (

Table 2. Comparison between plasma CTSB and NHL subtype.

	High-Grade NHL, n = 18	Low-Grade NHL, n = 26	p
CTSB (ng/mL)	14.27 (11.65–15.62)	15.99 (11.64–18.67)	0.122

Compared by Mann-Whitney U test, Abbreviations: CTSB, cathepsin B; NHL, non-Hodgkin’s lymphoma; p, level of significance.

).

3.4. Plasma CTSB mRNA Levels and CTSB Levels in Non-Hodgkin Lymphoma Patients

In addition, we monitored mRNA levels in plasma as a novel analytical method for non-invasive in vivo evaluation and surveillance of gene expression profiles in lymphomas. Figure 2 shows the plasma CTSB mRNA levels and protein CTSB levels for 18 patients before and after therapy. There was no significant difference in these parameters before and after therapy, although the median CTSB protein level was 12.15 (11.7–15.96) before therapy and 14.58 (11.11–17.96) after therapy. The medians for CTSB mRNA were very similar before and after therapy, 0.079 (0.037–0.095) vs. 0.065 (0.026–0.098).

3.5. The Correlation of Plasma CTSB with the CTSB mRNA Level and Redox Parameters in NHL Patients Before and After Therapy

We investigated the correlation of CTSB with CTSB mRNA, redox biomarkers and Cys C levels before and after therapy. Prior to therapy, plasma CTSB protein levels showed no correlation with CTSB mRNA levels, redox parameters or Cys C. However, after therapy, a positive association was observed between plasma CTSB protein levels and CTSB mRNA levels. Additionally, the TOS exhibited a positive correlation, while TAS and Cys C showed a negative correlation with plasma CTSB protein levels. Moreover, a significant correlation was identified between CTSB levels measured before and after therapy (r = 0.577; p = 0.012). These findings are summarized in

Table 3. The significant correlations of CTSB with CTSB mRNA, redox parameters and Cys C after therapy.

	CTSB mRNA r (p)	TAS mmol/L r (p)	TOS mmol/L r (p)	Cys C mg/L r (p)
CTSB (ng/mL) n = 18	0.591 (0.026)	−0.499 (0.035)	0.576 (0.012)	−0.687 (0.002)

r, Spearman’s correlation coefficient; p, level of significance; CTSB, cathepsin B; TOS, total oxidant status; TAS, total antioxidant status; Cys C, cystatin C.

.

A rank-based regression was performed in order to explore the sensitivity of the observed correlations to treatment-induced changes. The results support the findings from Spearman’s correlations and provide further evidence that CTSB mRNA and redox parameters may be meaningfully associated with CTSB protein levels after treatment (Table S1).

4. Discussion

NHL comprises a diverse array of malignancies distinguished by unique biological characteristics, clinical presentations and prognostic implications [16]. However, understanding the molecular pathways implicated in NHL etiology can facilitate the discovery of novel biomarkers and the development of targeted therapies.

Several lines of in vivo [23,24] and in vitro studies [25] support a role for CTSB in lymphoproliferative disorders. As previously stated, this study found higher plasma CTSB levels among NHL patients compared to healthy controls. Although it still remains unclear whether the elevated CTSB in plasma is derived from tumor cells or other stromal sources, it is established that malignant B cells can produce and secrete CTSB. The product of CTSB transcript was found in the extracellular matrix, suggesting enzyme release from proliferating or necrotic cells, especially from the cells growing under acidic pericellular conditions [7,26]. In addition, the elevation of CTSB concentration was shown in the sera of patients with some tumor types, including nasopharyngeal carcinoma and bladder cancer [27,28]. Although, it was shown that a higher concentration or increased proteolytic activity of CTSB was linked with an advancing tumor grade [29], our results showed that the concentration of CTSB was not significantly associated with NHL subtype.

The next step of this research was assessing the CTSB mRNA. To the best of our knowledge, there are no previous studies of CTSB mRNA levels in plasma among individuals with non-Hodgkin’s lymphoma, although CTSB mRNA overexpression has been observed in one research including individuals with leukemia [23]. As aforementioned, the mRNA levels in plasma were monitored in plasma before and after therapy and there is no statistically significant difference between CTSB mRNA before and after therapy. Despite the fact that results showed a lack of correlation in CTSB and CTSB mRNA levels before therapy, a significant correlation between CTSB levels in plasma and CTSB mRNA following therapy was observed. Moreover, our results are in concordance with other studies which confirmed a link between the levels of CTSB mRNA, protein and activity and tumor growth in certain malignancies [17,30,31]. The possible explanation lies in the an imbalance between the level of protein and level of CTSB mRNA in a cancer cell, which may represent a reliable disease progression marker [32]. A positive correlation observed after therapy should be elucidated through the attainment of equilibrium between protein synthesis and gene expression following the intervention. mRNA expression can be rapid and transient, while proteins remain in circulation for a longer period, as we demonstrated in Figure 2. Increased cathepsin B protein levels without changes in mRNA levels might be the result of S-nitrosylation, a post-translational modification that enhances its protein expression by stabilizing its own mRNA through adenosine-to-inosine (A-to-I) RNA editing or stress-induced regulation because oxidative stress may trigger mechanisms such as S-nitrosylation, or altered trafficking that increases cathepsin B protein levels independently of mRNA changes [33,34]. However, the regulation of CTSB at both the transcriptional and post-transcriptional stages remains inadequately comprehended. Upon gaining a comprehensive understanding of the various regulatory mechanisms governing CTSB expression and activity, novel techniques may emerge to selectively diminish CTSB activity in malignancies.

In this work, we analyzed the levels of redox markers in both the control group and the NHL patient group, along with their relationships with CTSB. The results showed that TAS level was significantly decreased; however, TOS concentrations were markedly elevated in the NHL group relative to the control group. Consistent with the findings described here, many studies have demonstrated that oxidative stress (OS) might overwhelm cellular defenses, hence facilitating programmed apoptotic cell death and lymphoid malignant development [4,35,36]. Chemotherapy can change the levels of oxidative stress and antioxidants in patients with cancer [37]. Taherkhani et al. have shown that a concurrent significant increase in malondialdehyde levels and a significant decrease in TAS levels were observed after three cycles of chemotherapy in patients with breast cancer [38]. However, in this study, we assessed the associations between TAS and TOS with the CTSB level. Prior to medication, we did not notice a link between CTSB and redox measures; this may account for the observed phenomenon that cancer cells are characteristic of uncontrolled cell proliferation and ROS production [39]. On the other hand, our study has shown that TOS had a positive association, while TAS demonstrated a negative correlation with the level of CTSB after therapy.

Both cathepsins and oxidative stress can mutually influence each other’s activity and effects. For example, oxidative stress can influence the subcellular localization of cathepsins. Under normal conditions, cathepsins are predominantly localized within lysosomes, performing their proteolytic functions. However, oxidative stress can disrupt the integrity of lysosomes, causing their rupture and leading to the release of cathepsins into the cytosol, where they can contribute to pathological processes, such as apoptosis and inflammation [8,35,40], which could undoubtedly elucidate our findings. The lack of correlation between CTSB and parameters of oxidative stress, as well as its natural inhibitor Cys C, before therapy indicates profound systemic disturbances caused by malignancy, where the parameters act independently due to the complexity of the tumor microenvironment and disrupted physiological functions [37].

Finally, we observed significantly higher concentrations of cystatin C in our NHL patients compared to the controls. The involvement of Cys C has been suggested with the alteration of the proteolytic system in cancer. The observation that Cys C levels were elevated in NHL patients compared to healthy individuals aligns with other research [41,42]. Cystatins efficiently block a limited number of catalytically active proteases, including CTSB. Consequently, this knowledge may elucidate associations between these two parameters [43].

The negative correlation between CTSB and Cys C in the context of NHL may be significant for understanding the pathogenesis and progression of the disease, as well as for potential prognostic or therapeutic approaches. CTSB is associated with the degradation of ECM and the promotion of the invasive potential of tumor cells. When CTSB levels increase, Cys C levels often decrease, either due to enhanced degradation, depletion of the inhibitor, or regulatory disruptions within the tumor microenvironment. Thus, reduced Cys C levels could indicate insufficient inhibition of CTSB, facilitating disease progression.

One important limitation in our study is the small number of patients. The cohort of patients observed post-therapy is notably limited. The study was undertaken during the COVID-19 pandemic, with fatalities from the virus and travel restrictions being the primary reasons for patients’ inability to return post-therapy. Therefore, future studies with a larger number of participants will provide valuable additional information.

5. Conclusions

This pilot study found that plasma CTSB levels are significantly altered in NHL patients compared to healthy controls, suggesting that CTSB can potentially contribute to the diagnostic process of NHL. However, no significant difference in CTSB levels was observed between high-grade and low-grade NHL, suggesting it may not correlate with lymphoma grade. Post-therapy, a positive correlation was found between CTSB protein and mRNA levels, and CTSB protein levels exhibited significant correlations with redox markers. Further studies involving a significantly larger number of patients may build upon our initial findings regarding the complex mechanisms underlying NHL development, potentially guiding the development of effective treatment strategies.